Targeted deletion of cellular dna sequences

ABSTRACT

Disclosed herein are methods and compositions for targeted deletion of double-stranded DNA. The compositions include fusion proteins comprising a cleavage domain (or cleavage half-domain) and an engineered zinc finger domain, and polynucleotides encoding same. Methods for targeted deletion include introduction of such fusion proteins, or polynucleotides encoding same, into a cell such that two targeted cleavage events occur. Subsequent cellular repair mechanisms result in deletion of sequences between the two cleavage sites.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.15/610,262, filed May 31, 2017, which is a continuation of U.S. patentapplication Ser. No. 13/784,634, filed Mar. 4, 2013, now U.S. Pat. No.9,695,442, which is a continuation of U.S. patent application Ser. No.11/304,981, filed Dec. 15, 2005, now U.S. Pat. No. 8,409,861, which iscontinuation-in-part of U.S. patent application Ser. No. 10/912,932,filed Aug. 6, 2004, now U.S. Pat. No. 7,888,121, and claims the benefitof U.S. Provisional Application No. 60/649,515, filed Feb. 3, 2005. U.S.application Ser. No. 10/912,932 also claims the benefit of the followingU.S. provisional patent applications: 60/493,931, filed Aug. 8, 2003;60/518,253, filed Nov. 7, 2003; 60/530,541, filed Dec. 18, 2003;60/542,780, filed Feb. 5, 2004; 60/556,831 filed Mar. 26, 2004; and60/575,919, filed Jun. 1, 2004. The disclosures of all of the above arehereby incorporated by reference in their entireties for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 24, 2020, isnamed 8325_0036_10_SL.txt and is 12,793 bytes in size.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

Not applicable.

TECHNICAL FIELD

The present disclosure is in the field of genome engineering andtargeted deletion (i.e. “knock-out” technology).

BACKGROUND

A major area of interest in genome biology, especially in light of thedetermination of the complete nucleotide sequences of a number ofgenomes, is the targeted alteration of genome sequences. One suchalteration is deletion, i.e., removal of sequences from a genome.Deletions can be as small as a single nucleotide pair, or can encompasshundreds, thousands or even millions of nucleotide pairs. The ability toreproducibly induce targeted deletions is useful in the identificationof gene function (e.g., by gene “knock-out” studies) and can also beuseful for inactivating genes (e.g., viral receptors) whose function isrequired for pathological processes.

Induction of small deletions by targeted cleavage of chromosomal DNAusing zinc finger/nuclease fusion proteins (ZFNs) has been described.See, for example, International Patent Publication No. WO 03/87341 andU.S. Patent Publication No. 2005/0064474. In brief, when a ZFN dimer (orany site-specific nuclease) is expressed within a cell, the process oftargeted cleavage, followed by error-prone repair, can lead togeneration of deletions, most of which are of fewer than about 20 bases,at or near the site of nuclease cleavage.

The process of ZFN-mediated mutagenesis as currently implemented using asingle ZFN dimer has a number of limitations. First, the sizes of thedeletions introduced by this method are generally quite small. Althoughdeletions in excess of 100 bp are occasionally seen, the vast majorityof deletions (probably more than 90%) are of fewer than about 20 bp.Therefore the method is unsuitable for generating large deletions athigh efficiency. The ability to generate large deletions at highfrequency would be required if, for example, it were necessary toeliminate entire regulatory region of a gene.

A second shortcoming of existing methods for ZFN-mediated mutagenesis isthat the heterogeneity of the deletions, coupled with their small sizes,makes it extremely difficult to monitor or quantify the mutagenesisprocess using conventional approaches such as PCR. By contrast, largerdeletions are much more readily detected and quantified in a backgroundof excess unmodified gene sequence using a standard method such as PCRfollowed by agarose gel electrophoresis.

Thus, methods for reproducibly obtaining large deletions of chromosomalsequence at high frequency would be useful in a number of areas ofgenome biology.

SUMMARY

The present disclosure provides compositions and methods for targetedmutagenesis, particularly deletion mutagenesis, of double-stranded DNAsequences. Thus, in one embodiment, a method for deleting sequences in aregion of interest in double-stranded DNA is provided, the methodcomprising expressing first, second, third and fourth fusion proteins ina cell, wherein each of the fusion proteins comprises (i) a zinc fingerDNA-binding domain that binds to a target site in the DNA, and (ii) acleavage half-domain; further wherein (a) the first and second fusionproteins bind to first and second target sites respectively, wherein afirst cleavage site lies between the first and second target sites(i.e., the first and second target sites straddle the first cleavagesite) and (b) the third and fourth fusion proteins bind to third andfourth target sites respectively, wherein a second cleavage site liesbetween the third and fourth target sites (i.e., the third and fourthtarget sites straddle the second cleavage site); such that the first andsecond fusion proteins cleave the DNA at the first cleavage site, thethird and fourth fusion proteins cleave the DNA at the second cleavagesite, and DNA ends are rejoined such that sequences between the firstand second cleavage sites are deleted.

Also provided is a method for deleting sequences in a region of interestin double-stranded DNA, the method comprising expressing first andsecond nucleases in a cell, wherein the first nuclease cleaves a firstcleavage site and the second nuclease cleaves a second cleavage site;and DNA ends are rejoined such that sequences between the first andsecond cleavage sites are deleted.

In certain embodiments, at least one of the nucleases is a fusionprotein comprising (i) a zinc finger DNA-binding domain that binds to atarget site in the DNA, wherein the target site is at or adjacent to thefirst or second cleavage site; and (ii) a cleavage domain.

Four DNA ends are generated by cleavage at the two cleavage sites. Firstand second DNA ends are generated by cleavage at the first cleavagesite; while third and fourth DNA ends are generated by cleavage at thesecond cleavage site. In certain embodiments, the first and secondcleavage sites are present on the same DNA molecule (e.g., on the samechromosome). In these cases, if the second and third ends, as definedabove, are considered to be part of a DNA fragment containing sequencesthat lie between the first and second cleavage sites (i.e. a fragmentthat is released by cleavage at the first and second cleavage sites),then rejoining of the first and fourth ends results in deletion ofsequences between the first and second cleavage sites. An alternativeoutcome is inversion of some or all of the sequences located between thefirst and second cleavage sites.

If the first and second cleavage sites are located on differentchromosomes, chromosomal translocations and/or chromosomal fusions canresult. Finally, targeted cleavages and resultant deletion can alsooccur on extrachromosomal nucleic acids, such as episomes, intracellularvectors, organellar genomes, etc.

In certain instances, ends generated directly by the cleavage event(e.g., the first and fourth DNA ends) may be rejoined to cause adeletion. In other instances, the ends generated by cleavage may befurther processed (e.g., by exonucleolytic resection) and these endsresulting from cleavage can be rejoined. Rejoining can occur by cellularrepair mechanisms such as those collectively denoted “non-homologousend-joining.”

As described above, in certain embodiments, sequences in a region ofinterest are deleted, wherein the region of interest is in cellularchromatin. In these cases, the first and second cleavage sites can be onthe same chromosomes, on different chromosomes, on an extrachromosomalnucleic acid, or the first cleavage sit can be present on a chromosomeand the second cleavage site can be present on an extrachromosomalnucleic acid.

The target sites bound by the fusion proteins are present in pairswherein, for each pair of target sites, a cleavage site liestherebetween. Thus, the first and second target sites straddle a firstcleavage site and the third and fourth target sites straddle a secondcleavage site. The target sites can be separated by any number ofnucleotide pairs, commensurate with dimerization of the fusion proteinsto regenerate a functional cleavage domain. As described elsewhere inthis disclosure, maximal cleavage efficiency varies with both thedistance between target sites and the length of the linker sequencesbetween the zinc finger portion and the nuclease half-domain portion ofthe fusion proteins. Accordingly, the first and second target sites canbe separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide pairs.Similarly, the third and fourth target sites can be separated by 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more nucleotide pairs. When discussing thedistance between target sites, this distance is expressed as the numberof nucleotide pairs intervening between the near edges of the targetsites, and does not include any nucleotide pair that is present ineither of the target sites.

The size of a deletion induced by the disclosed methods and compositionsis determined by the distance between the first and second cleavagesites. Accordingly, deletions of any size, in any region of interest,can be obtained. Deletions of 25, 50, 100, 200, 300, 400, 500, 600, 700,800, 900, 1,000 nucleotide pairs, or any integral value of nucleotidepairs within this range, can be obtained. In addition deletions of asequence of any integral value of nucleotide pairs greater than 1,000nucleotide pairs can be obtained using the methods and compositionsdisclosed herein.

The region of interest, in which deletion is induced, can be in a gene.The gene can be a gene involved in a disease or pathological condition.For example, the gene can be a viral receptor. Certain chemokinereceptors also function as viral receptors; for example, the chemokinereceptor CCR-5 also functions a receptor for human immunodeficiencyvirus (HIV), the causative agent of acquired immune deficiency syndrome(AIDS). Thus, the present disclosure provides methods for inducingtargeted deletions in a CCR-5 gene, optionally a human CCR-5 gene, fortreatment of AIDS.

Also provided are deleted CCR-5 gene sequences and cells comprisingdeleted CCR-5 genes; optionally, human cells. In certain embodiments,the cells are primary cells obtained from an individual, which mayoptionally be returned to the same individual or a different individual.In certain embodiments, the primary cells are T-cells or dendriticcells.

Cells can also include cultured cells, cells in an organism and cellsthat have been removed from an organism for treatment in cases where thecells and/or their descendants will be returned to the organism aftertreatment. A region of interest in cellular chromatin can be, forexample, a genomic sequence or portion thereof. Compositions includefusion polypeptides comprising an engineered zinc finger binding domain(e.g., a zinc finger binding domain having a novel specificity) and acleavage domain, and fusion polypeptides comprising an engineered zincfinger binding domain and a cleavage half-domain. Cleavage domains andcleavage half domains can be obtained, for example, from variousrestriction endonucleases and/or homing endonucleases.

Cellular chromatin can be present in any type of cell including, but notlimited to, prokaryotic and eukaryotic cells, fungal cells, plant cells,animal cells, mammalian cells, primate cells and human cells.

A protein e.g., a fusion protein, can be expressed in a cell, e.g., bydelivering the fusion protein to the cell or by delivering apolynucleotide encoding the fusion protein to a cell, wherein thepolynucleotide, if DNA, is transcribed, and an RNA molecule delivered tothe cell or a transcript of a DNA molecule delivered to the cell istranslated, to generate the protein. Methods for polynucleotide andpolypeptide delivery to cells are presented elsewhere in thisdisclosure.

In the disclosed methods for targeted deletion, the cleavagehalf-domains can be derived from the same endonuclease or from differentendonucleases. Endonucleases include, but are not limited to, homingendonucleases and restriction endonucleases. Exemplary restrictionendonucleases are Type IIS restriction endonucleases; an exemplary TypeIIS restriction endonuclease is FokI.

In certain embodiments, a cleavage domain can comprise two cleavagehalf-domains that are covalently linked in the same polypeptide. The twocleavage half-domains can be derived from the same endonuclease or fromdifferent endonucleases. The cleavage half domain can be derived from,for example, a homing endonuclease or a restriction endonuclease, forexample, a Type IIS restriction endonuclease. An exemplary Type IISrestriction endonuclease is FokI.

In certain embodiments, it is possible to obtain increased cleavagespecificity by utilizing fusion proteins in which one or both cleavagehalf-domains contains an alteration in the amino acid sequence of thedimerization interface.

In the aforementioned methods for targeted deletion, the target sitesfor the fusion proteins can comprise any number of nucleotides.Preferably, they are at least nine nucleotides in length, but they canalso be larger (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18 and up to 100nucleotides, including any integral value between 9 and 100nucleotides); moreover, different target sequences need not necessarilybe the same length. The distance between the nearest edges of the targetsites can be any integral number of nucleotide pairs between 1 and 50,(e.g., 5 or 6 base pairs) as measured from the near end of one bindingsite to the closest end of the other binding site.

In the aforementioned methods for targeted deletion, cellular chromatincan be cleaved at a site located between the target sites of two fusionproteins. In certain embodiments, the target sites are on opposite DNAstrands. Moreover, expression of the fusion proteins in the cell can beaccomplished either by introduction of the proteins into the cell or byintroduction of one or more polynucleotides into the cell, which areoptionally transcribed (if the polynucleotide is DNA), and thetranscript(s) translated, to produce the fusion proteins. For example,two polynucleotides, each comprising sequences encoding one pair offusion proteins, can be introduced into a cell. Alternatively, a singlepolynucleotide comprising sequences encoding both pairs of fusionproteins can be introduced into the cell.

In any of the methods described herein, a zinc finger binding domain canbe engineered, for example designed and/or selected. See, for example,U.S. Pat. Nos. 5,789,538; 6,007,988; 6,013,453; 6,140,466; 6,242,568;6,410,248; 6,453,242; 6,534,261; 6,733,970; 6,746,838; 6,785,613;6,790,941; 6,794,136; 6,866,997; and 6,933,113, as well as U.S. PatentPublication No. 2005/0064477. See also International Patent PublicationNo. WO 02/42459.

Polynucleotides encoding fusions between a zinc finger binding domainand a cleavage domain or cleavage half-domain can be DNA or RNA, can belinear or circular, and can be single-stranded or double-stranded. Theycan be delivered to the cell as naked nucleic acid, as a complex withone or more delivery agents (e.g., liposomes, poloxamers) or containedin a viral delivery vehicle, such as, for example, an adenovirus,adeno-associated virus (AAV) or lentivirus. A polynucleotide can encodeone or more fusion proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of an agarose gel in which amplification productsof the human CCR-5 gene were analyzed. Lane 1 shows amplificationproducts of DNA from K562 cells transfected with a plasmid encodinggreen fluorescent protein. Lane 2 shows amplification products of DNAfrom K562 cells transfected with a plasmid encoding a pair of ZFNsdesigned to cleave between +162 and +168 of the human CCR-5 gene. Lane 3shows amplification products of DNA from K562 cells transfected with aplasmid encoding a pair of ZFNs designed to cleave between +627 and +633of the human CCR-5 gene. Lane 4 shows amplification products of DNA fromK562 cells transfected with both of the aforementioned ZFN pairs.

FIG. 2 shows nucleotide sequences of amplification products of the CCR-5gene obtained from K562 cells that had been transfected with twoplasmids: one encoding a pair of ZFNs designed to cleave between +162and +168 of the human CCR-5 gene and the other encoding a pair of ZFNsdesigned to cleave between +627 and +633 of the human CCR-5 gene (i.e.,corresponding to lane 4 of FIG. 1). The topmost line shows a partialnucleotide sequence of the wild-type human CCR-5 gene from +146 to +185(SEQ ID NO:45) and from +616 to +646 (SEQ ID NO:46), with the ZFN targetsites underlined. Coordinates are with respect to the first nucleotidepair of the translation initiation codon.

Each of the remaining lines (SEQ ID NOS:47-62) represents a contiguousnucleotide sequence obtained from an amplification product of DNAobtained from cells that had been transfected as described in thepreceding paragraph. In the written representation shown in the Figure,portions of each contiguous sequence have been separated for purposes ofalignment with the wild-type sequence. The bottom-most two lines providedescriptions of two additional sequences that were obtained.

FIG. 3 shows an autoradiogram of a Southern blot of genomic DNA purifiedfrom transfected K562 cells and digested with XhoI and NdeI. Lane 1shows DNA from cells transfected with two plasmids: one encoding a pairof ZFNs designed to cleave between +162 and +168 of the human CCR-5 geneand the other encoding a pair of ZFNs designed to cleave between +627and +633 of the human CCR-5 gene. Lane 2 shows DNA from cellstransfected with a plasmid encoding GFP. The upper arrow indicates aband derived from amplification of wild-type sequences; the lower arrowidentifies a band obtained from amplification of deleted CCR-5 loci.

A schematic diagram of a portion of the human CCR-5 gene, indicatingrelevant restriction enzyme recognition sites, ZFN target sites and theapproximate map position of the fragment used as probe, is shown belowthe autoradiogram. Numbering is with respect to the first nucleotidepair of the initiation codon.

FIG. 4 is a photograph of an agarose gel in which amplification productsof the human CCR-5 gene were analyzed. Lane 1 shows size markers. Lane 2shows amplification products of DNA from human T-cells transfected witha plasmid encoding a pair of ZFNs designed to cleave between +162 and+168 of the human CCR-5 gene and a plasmid encoding a pair of ZFNsdesigned to cleave between +627 and +633 of the human CCR-5 gene. Lane 3shows amplification products of DNA from human T-cells transfected witha plasmid encoding green fluorescent protein. The arrow indicates a bandrepresenting amplification products of deleted CCR-5 loci.

FIG. 5 shows nucleotide sequences of amplification products of the CCR-5gene obtained from primary human T-cells that had been transfected withtwo plasmids: one encoding a pair of ZFNs designed to cleave between+162 and +168 of the human CCR-5 gene and the other encoding a pair ofZFNs designed to cleave between +627 and +633 of the human CCR-5 gene.The topmost line shows a partial nucleotide sequence of the wild-typehuman CCR-5 gene from +146 to +185 (SEQ ID NO: 63) and from +616 to +645(SEQ ID NO: 64). The target sites for the ZFNs are underlined.Coordinates are with respect to the first nucleotide pair of thetranslation initiation codon.

Each of the remaining lines (SEQ ID NOS:65-84) represents a contiguousnucleotide sequence obtained from an amplification product of DNAobtained from cells that had been transfected as described in thepreceding paragraph. In the written representation shown in the Figure,portions of each contiguous sequence have been separated for purposes ofalignment with the wild-type sequence.

DETAILED DESCRIPTION

Disclosed herein are compositions and methods useful for targeteddeletion of sequences in double-stranded DNA (e.g., cellular chromatin).Double-stranded DNA includes that present in chromosomes, episomes,organellar genomes (e.g., mitochondria, chloroplasts), artificialchromosomes and any other type of nucleic acid present in a cell suchas, for example, amplified sequences, double minute chromosomes and thegenomes of endogenous or infecting bacteria and viruses. Chromosomalsequences can be normal (i.e., wild-type) or mutant; mutant sequencescan comprise, for example, insertions, deletions, translocations,rearrangements, and/or point mutations. A chromosomal sequence can alsocomprise one of a number of different alleles.

Compositions useful for targeted deletion include pairs of fusionproteins, each fusion protein comprising a cleavage domain (or acleavage half-domain) and a zinc finger binding domain, polynucleotidesencoding these proteins and combinations of polypeptides andpolypeptide-encoding polynucleotides. A zinc finger binding domain cancomprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or morezinc fingers), and can be engineered to bind to any sequence. Thus, byidentifying a target genomic region, the deletion of which is desired,one can, according to the methods disclosed herein, construct one ormore fusion proteins comprising a cleavage domain (or cleavagehalf-domain) and a zinc finger domain engineered to recognize a targetsequence in said genomic region. The presence of such fusion proteins ina cell results in binding of the fusion proteins to their target sites,cleavage at two cleavage sites, and deletion of sequences therebetween.

Thus, to obtain targeted deletion, cells are treated simultaneously witha pair of ZFN dimers, which stimulates the highly efficient deletion ofDNA lying between the sites cleaved by the two dimers. In this context,the term “ZFN dimer” refers to a pair of zinc finger/cleavage halfdomain fusion proteins, each of which binds to a distinct target sitesuch that DNA is cleaved at a cleavage site which lies between thetarget sites.

Advantages of the methods for targeted deletion mutagenesis disclosedherein include:

1) The process can be used for highly efficient deletion of large DNAsequences (e.g., several hundred base pairs). This enables disruption ofDNA elements (e.g., exons, introns, regulatory sequences) that may notbe completely removable via introduction of small (<20 bp) deletions.

2) The process is readily monitored using standard molecular biologymethods such as PCR followed by agarose gel electrophoresis, as well asSouthern blot analysis.

3) The induction of large deletions occurs at an efficiency that issubstantially enhanced relative to the mutational efficiencies achievedby cleavage with either of the individual ZFN dimers used in theprocess. Thus use of a pair of ZFN dimers provides a general means forachieving deletion efficiencies which are higher than those achievableusing a single ZFN dimer.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

Definitions

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of a correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶M⁻¹ orlower. “Affinity” refers to the strength of binding: increased bindingaffinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

Zinc finger binding domains can be “engineered” to bind to apredetermined nucleotide sequence. Non-limiting examples of methods forengineering zinc finger proteins are design and selection. A designedzinc finger protein is a protein not occurring in nature whosedesign/composition results principally from rational criteria. Rationalcriteria for design include application of substitution rules andcomputerized algorithms for processing information in a database storinginformation of existing ZFP designs and binding data. See, for example,U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see alsoInternational Patent Publication Nos. WO 98/53058; WO 98/53059; WO98/53060; WO 02/016536; and WO 03/016496.

A “selected” zinc finger protein is a protein not found in nature whoseproduction results primarily from an empirical process such as phagedisplay, interaction trap or hybrid selection. See e.g., U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; and 6,200,759; andInternational Patent Publication Nos. WO 95/19431; WO 96/06166; WO98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197; and WO02/099084.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “homologous, non-identical sequence” refers to a first sequence whichshares a degree of sequence identity with a second sequence, but whosesequence is not identical to that of the second sequence. For example, apolynucleotide comprising the wild-type sequence of a mutant gene ishomologous and non-identical to the sequence of the mutant gene. Incertain embodiments, the degree of homology between the two sequences issufficient to allow homologous recombination therebetween, utilizingnormal cellular mechanisms. Two homologous non-identical sequences canbe any length and their degree of non-homology can be as small as asingle nucleotide (e.g., for correction of a genomic point mutation bytargeted homologous recombination) or as large as 10 or more kilobases(e.g., for insertion of a gene at a predetermined ectopic site in achromosome). Two polynucleotides comprising the homologous non-identicalsequences need not be the same length. For example, an exogenouspolynucleotide (i.e., donor polynucleotide) of between 20 and 10,000nucleotides or nucleotide pairs can be used.

Techniques for determining nucleic acid and amino acid sequence identityare known in the art. Typically, such techniques include determining thenucleotide sequence of the mRNA for a gene and/or determining the aminoacid sequence encoded thereby, and comparing these sequences to a secondnucleotide or amino acid sequence. Genomic sequences can also bedetermined and compared in this fashion. In general, identity refers toan exact nucleotide-to-nucleotide or amino acid-to-amino acidcorrespondence of two polynucleotides or polypeptide sequences,respectively. Two or more sequences (polynucleotide or amino acid) canbe compared by determining their percent identity. The percent identityof two sequences, whether nucleic acid or amino acid sequences, is thenumber of exact matches between two aligned sequences divided by thelength of the shorter sequences and multiplied by 100. An approximatealignment for nucleic acid sequences is provided by the local homologyalgorithm of Smith and Waterman (1981) Advances in Applied Mathematics2:482-489. This algorithm can be applied to amino acid sequences byusing the scoring matrix developed by Dayhoff, Atlas of ProteinSequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, NationalBiomedical Research Foundation, Washington, D.C., USA, and normalized byGribskov (1986) Nucl. Acids Res. 14(6):6745-6763. An exemplaryimplementation of this algorithm to determine percent identity of asequence is provided by the Genetics Computer Group (Madison, Wis.) inthe “BestFit” utility application. The default parameters for thismethod are described in the Wisconsin Sequence Analysis Package ProgramManual, Version 8 (1995) (available from Genetics Computer Group,Madison, Wis.). A preferred method of establishing percent identity inthe context of the present disclosure is to use the MPSRCH package ofprograms copyrighted by the University of Edinburgh, developed by JohnF. Collins and Shane S. Sturrok, and distributed by IntelliGenetics,Inc. (Mountain View, Calif.). From this suite of packages theSmith-Waterman algorithm can be employed where default parameters areused for the scoring table (for example, gap open penalty of 12, gapextension penalty of one, and a gap of six). From the data generated the“Match” value reflects sequence identity. Other suitable programs forcalculating the percent identity or similarity between sequences aregenerally known in the art, for example, another alignment program isBLAST, used with default parameters. Details of these programs can befound online. With respect to sequences described herein, the range ofdesired degrees of sequence identity is approximately 80% to 100% andany integer value therebetween. Typically the percent identities betweensequences are at least 70-75%, preferably 80-82%, more preferably85-90%, even more preferably 92%, still more preferably 95%, and mostpreferably 98% sequence identity.

Alternatively, the degree of sequence similarity between polynucleotidescan be determined by hybridization of polynucleotides under conditionsthat allow formation of stable duplexes between homologous regions,followed by digestion with single-stranded-specific nuclease(s), andsize determination of the digested fragments. Two nucleic acid, or twopolypeptide sequences are substantially homologous to each other whenthe sequences exhibit at least about 70%-75%, preferably 80%-82%, morepreferably 85%-90%, even more preferably 92%, still more preferably 95%,and most preferably 98% sequence identity over a defined length of themolecules, as determined using the methods above. As used herein,substantially homologous also refers to sequences showing completeidentity to a specified DNA or polypeptide sequence. DNA sequences thatare substantially homologous can be identified in a Southernhybridization experiment under, for example, stringent conditions, asdefined for that particular system. Defining appropriate hybridizationconditions is within the skill of the art. See, e.g., Sambrook et al.,supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D.Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

Selective hybridization of two nucleic acid fragments can be determinedas follows. The degree of sequence identity between two nucleic acidmolecules affects the efficiency and strength of hybridization eventsbetween such molecules. A partially identical nucleic acid sequence willat least partially inhibit the hybridization of a completely identicalsequence to a target molecule. Inhibition of hybridization of thecompletely identical sequence can be assessed using hybridization assaysthat are well known in the art (e.g., Southern (DNA) blot, Northern(RNA) blot, solution hybridization, or the like, see Sambrook, et al.,Molecular Cloning: A Laboratory Manual, Second Edition, (1989) ColdSpring Harbor, N.Y.). Such assays can be conducted using varying degreesof selectivity, for example, using conditions varying from low to highstringency. If conditions of low stringency are employed, the absence ofnon-specific binding can be assessed using a secondary probe that lackseven a partial degree of sequence identity (for example, a probe havingless than about 30% sequence identity with the target molecule), suchthat, in the absence of non-specific binding events, the secondary probewill not hybridize to the target.

When utilizing a hybridization-based detection system, a nucleic acidprobe is chosen that is complementary to a reference nucleic acidsequence, and then by selection of appropriate conditions the probe andthe reference sequence selectively hybridize, or bind, to each other toform a duplex molecule. A nucleic acid molecule that is capable ofhybridizing selectively to a reference sequence under moderatelystringent hybridization conditions typically hybridizes under conditionsthat allow detection of a target nucleic acid sequence of at least about10-14 nucleotides in length having at least approximately 70% sequenceidentity with the sequence of the selected nucleic acid probe. Stringenthybridization conditions typically allow detection of target nucleicacid sequences of at least about 10-14 nucleotides in length having asequence identity of greater than about 90-95% with the sequence of theselected nucleic acid probe. Hybridization conditions useful forprobe/reference sequence hybridization, where the probe and referencesequence have a specific degree of sequence identity, can be determinedas is known in the art (see, for example, Nucleic Acid Hybridization: APractical Approach, editors B. D. Hames and S. J. Higgins, (1985)Oxford; Washington, D.C.; IRL Press).

Conditions for hybridization are well-known to those of skill in theart. Hybridization stringency refers to the degree to whichhybridization conditions disfavor the formation of hybrids containingmismatched nucleotides, with higher stringency correlated with a lowertolerance for mismatched hybrids. Factors that affect the stringency ofhybridization are well-known to those of skill in the art and include,but are not limited to, temperature, pH, ionic strength, andconcentration of organic solvents such as, for example, formamide anddimethylsulfoxide. As is known to those of skill in the art,hybridization stringency is increased by higher temperatures, lowerionic strength and lower solvent concentrations.

With respect to stringency conditions for hybridization, it is wellknown in the art that numerous equivalent conditions can be employed toestablish a particular stringency by varying, for example, the followingfactors: the length and nature of the sequences, base composition of thevarious sequences, concentrations of salts and other hybridizationsolution components, the presence or absence of blocking agents in thehybridization solutions (e.g., dextran sulfate, and polyethyleneglycol), hybridization reaction temperature and time parameters, as wellas, varying wash conditions. The selection of a particular set ofhybridization conditions is selected following standard methods in theart (see, for example, Sambrook, et al., Molecular Cloning: A LaboratoryManual, Second Edition, (1989) Cold Spring Harbor, N.Y.).

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (FIR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells. This process requires nucleotide sequence homology,uses a “donor” molecule to template repair of a “target” molecule (i.e.,the one that experienced the double-strand break), and is variouslyknown as “non-crossover gene conversion” or “short tract geneconversion,” because it leads to the transfer of genetic informationfrom the donor to the target. Without wishing to be bound by anyparticular theory, such transfer can involve mismatch correction ofheteroduplex DNA that forms between the broken target and the donor,and/or “synthesis-dependent strand annealing,” in which the donor isused to resynthesize genetic information that will become part of thetarget, and/or related processes. Such specialized HR often results inan alteration of the sequence of the target molecule such that part orall of the sequence of the donor polynucleotide is incorporated into thetarget polynucleotide.

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage domain” comprises one or more polypeptide sequences whichpossesses catalytic activity for DNA cleavage. A cleavage domain can becontained in a single polypeptide chain or cleavage activity can resultfrom the association of two (or more) polypeptides.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity).

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome” is a chromatin complex comprising all or a portion of thegenome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

An “accessible region” is a site in cellular chromatin in which a targetsite present in the nucleic acid can be bound by an exogenous moleculewhich recognizes the target site. Without wishing to be bound by anyparticular theory, it is believed that an accessible region is one thatis not packaged into a nucleosomal structure. The distinct structure ofan accessible region can often be detected by its sensitivity tochemical and enzymatic probes, for example, nucleases.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist. For example, thesequence 5′-GAATTC-3′ is a target site for the Eco RI restrictionendonuclease.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPDNA-binding domain and a cleavage domain) and fusion nucleic acids (forexample, a nucleic acid encoding the fusion protein described supra).Examples of the second type of fusion molecule include, but are notlimited to, a fusion between a triplex-forming nucleic acid and apolypeptide, and a fusion between a minor groove binder and a nucleicacid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of a mRNA. Gene products also include RNAs whichare modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression.

“Eucaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFPDNA-binding domain is fused to a cleavage domain, the ZFP DNA-bindingdomain and the cleavage domain are in operative linkage if, in thefusion polypeptide, the ZFP DNA-binding domain portion is able to bindits target site and/or its binding site, while the cleavage domain isable to cleave DNA in the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain oneore more amino acid or nucleotide substitutions. Methods for determiningthe function of a nucleic acid (e.g., coding function, ability tohybridize to another nucleic acid) are well-known in the art. Similarly,methods for determining protein function are well-known. For example,the DNA-binding function of a polypeptide can be determined, forexample, by filter-binding, electrophoretic mobility-shift, orimmunoprecipitation assays. DNA cleavage can be assayed by gelelectrophoresis. See Ausubel et al., supra. The ability of a protein tointeract with another protein can be determined, for example, byco-immunoprecipitation, two-hybrid assays or complementation, bothgenetic and biochemical. See, for example, Fields et al. (1989) Nature340:245-246; U.S. Pat. No. 5,585,245 and International PatentPublication No. WO 98/44350.

Target Sites

The disclosed methods and compositions include fusion proteinscomprising a cleavage domain (or a cleavage half-domain) and a zincfinger domain, in which the zinc finger domain, by binding to a sequencein cellular chromatin (e.g., a target site or a binding site), directsthe activity of the cleavage domain (or cleavage half-domain) to thevicinity of the sequence and, hence, induces cleavage in the vicinity ofthe target sequence. As set forth elsewhere in this disclosure, a zincfinger domain can be engineered to bind to virtually any desiredsequence. Accordingly, after identifying a region of interest containinga sequence at which cleavage or recombination is desired, one or morezinc finger binding domains can be engineered to bind to one or moresequences in the region of interest. Expression of a fusion proteincomprising a zinc finger binding domain and a cleavage domain (or of twofusion proteins, each comprising a zinc finger binding domain and acleavage half-domain), in a cell, effects cleavage in the region ofinterest.

Selection of a sequence in cellular chromatin for binding by a zincfinger domain (e.g., a target site) can be accomplished, for example,according to the methods disclosed in co-owned U.S. Pat. No. 6,453,242(Sep. 17, 2002), which also discloses methods for designing ZFPs to bindto a selected sequence. It will be clear to those skilled in the artthat simple visual inspection of a nucleotide sequence can also be usedfor selection of a target site. Accordingly, any means for target siteselection can be used in the claimed methods.

Target sites are generally composed of a plurality of adjacent targetsubsites. A target subsite refers to the sequence (usually either anucleotide triplet, or a nucleotide quadruplet that can overlap by onenucleotide with an adjacent quadruplet) bound by an individual zincfinger. See, for example, International Patent Publication No. WO02/077227. If the strand with which a zinc finger protein makes mostcontacts is designated the target strand “primary recognition strand,”or “primary contact strand,” some zinc finger proteins bind to a threebase triplet in the target strand and a fourth base on the non-targetstrand. A target site generally has a length of at least 9 nucleotidesand, accordingly, is bound by a zinc finger binding domain comprising atleast three zinc fingers. However binding of, for example, a 4-fingerbinding domain to a 12-nucleotide target site, a 5-finger binding domainto a 15-nucleotide target site or a 6-finger binding domain to an18-nucleotide target site, is also possible. As will be apparent,binding of larger binding domains (e.g., 7-, 8-, 9-finger and more) tolonger target sites is also possible.

It is not necessary for a target site to be a multiple of threenucleotides. For example, in cases in which cross-strand interactionsoccur (see, e.g., U.S. Pat. No. 6,453,242 and International PatentPublication No. WO 02/077227), one or more of the individual zincfingers of a multi-finger binding domain can bind to overlappingquadruplet subsites. As a result, a three-finger protein can bind a10-nucleotide sequence, wherein the tenth nucleotide is part of aquadruplet bound by a terminal finger, a four-finger protein can bind a13-nucleotide sequence, wherein the thirteenth nucleotide is part of aquadruplet bound by a terminal finger, etc.

The length and nature of amino acid linker sequences between individualzinc fingers in a multi-finger binding domain also affects binding to atarget sequence. For example, the presence of a so-called “non-canonicallinker,” “long linker” or “structured linker” between adjacent zincfingers in a multi-finger binding domain can allow those fingers to bindsubsites which are not immediately adjacent. Non-limiting examples ofsuch linkers are described, for example, in U.S. Pat. No. 6,479,626 andInternational Patent Publication No. WO 01/53480. Accordingly, one ormore subsites, in a target site for a zinc finger binding domain, can beseparated from each other by 1, 2, 3, 4, 5 or more nucleotides. Toprovide but one example, a four-finger binding domain can bind to a13-nucleotide target site comprising, in sequence, two contiguous3-nucleotide subsites, an intervening nucleotide, and two contiguoustriplet subsites.

Distance between sequences (e.g., target sites) refers to the number ofnucleotides or nucleotide pairs intervening between two sequences, asmeasured from the edges of the sequences nearest each other.

In certain embodiments in which cleavage depends on the binding of twozinc finger domain/cleavage half-domain fusion molecules to separatetarget sites, the two target sites can be on opposite DNA strands. Inother embodiments, both target sites are on the same DNA strand.

Zinc Finger Binding Domains

A zinc finger binding domain comprises one or more zinc fingers. Milleret al. (1985) EMBO J. 4:1609-1614; Rhodes (1993) Scientific AmericanFebruary: 56-65; U.S. Pat. No. 6,453,242. Typically, a single zincfinger domain is about 30 amino acids in length. Structural studies havedemonstrated that each zinc finger domain (motif) contains two betasheets (held in a beta turn which contains the two invariant cysteineresidues) and an alpha helix (containing the two invariant histidineresidues), which are held in a particular conformation throughcoordination of a zinc atom by the two cysteines and the two histidines.

Zinc fingers include both canonical C₂H₂ zinc fingers (i.e., those inwhich the zinc ion is coordinated by two cysteine and two histidineresidues) and non-canonical zinc fingers such as, for example, C₃H zincfingers (those in which the zinc ion is coordinated by three cysteineresidues and one histidine residue) and C₄ zinc fingers (those in whichthe zinc ion is coordinated by four cysteine residues). See alsoInternational Patent Publication No. WO 02/057293.

Zinc finger binding domains can be engineered to bind to a sequence ofchoice. See, for example, Beerli et al. (2002) Nature Biotechnol.20:135-141; Pabo et al. (2001) Ann. Rev. Biochem. 70:313-340; Isalan etal. (2001) Nature Biotechnol. 19:656-660; Segal et al. (2001) Curr.Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr. Opin. Struct.Biol. 10:411-416. An engineered zinc finger binding domain can have anovel binding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, for example, co-owned U.S. Pat. Nos.6,453,242 and 6,534,261.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as International Patent Publication Nos. WO 98/37186; WO 98/53057;WO 00/27878; and WO 01/88197 and GB 2,338,237.

Enhancement of binding specificity for zinc finger binding domains hasbeen described, for example, in co-owned International PatentPublication No. WO 02/077227.

Since an individual zinc finger binds to a three-nucleotide (i.e.,triplet) sequence (or a four-nucleotide sequence which can overlap, byone nucleotide, with the four-nucleotide binding site of an adjacentzinc finger), the length of a sequence to which a zinc finger bindingdomain is engineered to bind (e.g., a target sequence) will determinethe number of zinc fingers in an engineered zinc finger binding domain.For example, for ZFPs in which the finger motifs do not bind tooverlapping subsites, a six-nucleotide target sequence is bound by atwo-finger binding domain; a nine-nucleotide target sequence is bound bya three-finger binding domain, etc. As noted herein, binding sites forindividual zinc fingers (i.e., subsites) in a target site need not becontiguous, but can be separated by one or several nucleotides,depending on the length and nature of the amino acids sequences betweenthe zinc fingers (i.e., the inter-finger linkers) in a multi-fingerbinding domain.

In a multi-finger zinc finger binding domain, adjacent zinc fingers canbe separated by amino acid linker sequences of approximately 5 aminoacids (so-called “canonical” inter-finger linkers) or, alternatively, byone or more non-canonical linkers. See, e.g., co-owned U.S. Pat. Nos.6,453,242 and 6,534,261. For engineered zinc finger binding domainscomprising more than three fingers, insertion of longer(“non-canonical”) inter-finger linkers between certain of the zincfingers may be preferred as it may increase the affinity and/orspecificity of binding by the binding domain. See, for example, U.S.Pat. No. 6,479,626 and International Patent Publication No. WO 01/53480.Accordingly, multi-finger zinc finger binding domains can also becharacterized with respect to the presence and location of non-canonicalinter-finger linkers. For example, a six-finger zinc finger bindingdomain comprising three fingers (joined by two canonical inter-fingerlinkers), a long linker and three additional fingers (joined by twocanonical inter-finger linkers) is denoted a 2×3 configuration.Similarly, a binding domain comprising two fingers (with a canonicallinker therebetween), a long linker and two additional fingers (joinedby a canonical linker) is denoted a 2×2 protein. A protein comprisingthree two-finger units (in each of which the two fingers are joined by acanonical linker), and in which each two-finger unit is joined to theadjacent two finger unit by a long linker, is referred to as a 3×2protein.

The presence of a long or non-canonical inter-finger linker between twoadjacent zinc fingers in a multi-finger binding domain often allows thetwo fingers to bind to subsites which are not immediately contiguous inthe target sequence. Accordingly, there can be gaps of one or morenucleotides between subsites in a target site; i.e., a target site cancontain one or more nucleotides that are not contacted by a zinc finger.For example, a 2×2 zinc finger binding domain can bind to twosix-nucleotide sequences separated by one nucleotide, i.e., it binds toa 13-nucleotide target site. See also Moore et al. (2001a) Proc. Natl.Acad. Sci. USA 98:1432-1436; Moore et al. (2001b) Proc. Natl. Acad. Sci.USA 98:1437-1441 and International Patent Publication No. WO 01/53480.

As mentioned previously, a target subsite is a three- or four-nucleotidesequence that is bound by a single zinc finger. For certain purposes, atwo-finger unit is denoted a binding module. A binding module can beobtained by, for example, selecting for two adjacent fingers in thecontext of a multi-finger protein (generally three fingers) which bind aparticular six-nucleotide target sequence. Alternatively, modules can beconstructed by assembly of individual zinc fingers. See alsoInternational Patent Publication Nos. WO 98/53057 and WO 01/53480.

Cleavage Domains

The cleavage domain portion of the fusion proteins disclosed herein canbe obtained from any endo- or exonuclease. Exemplary endonucleases fromwhich a cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., Si Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain (e.g., fusion proteins comprising azinc finger binding domain and a cleavage half-domain) can be derivedfrom any nuclease or portion thereof, as set forth above, that requiresdimerization for cleavage activity. In general, two fusion proteins arerequired for cleavage if the fusion proteins comprise cleavagehalf-domains. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins places the cleavage half-domains in aspatial orientation to each other that allows the cleavage half-domainsto form a functional cleavage domain, e.g., by dimerizing. Thus, incertain embodiments, the near edges of the target sites are separated by5-8 nucleotides or by 15-18 nucleotides. However any integral number ofnucleotides or nucleotide pairs can intervene between two target sites(e.g., from 2 to 50 nucleotides or more). In general, the point ofcleavage lies between the target sites.

In general, if two fusion proteins are used, each comprising a cleavagehalf-domain, the primary contact strand for the zinc finger portion ofeach fusion protein will be on a different DNA strands and in oppositeorientation. That is, for a pair of ZFP/cleavage half-domain fusions,the target sequences are on opposite strands and the two proteins bindin opposite orientation.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains. For example, the Type IIS enzyme FokI catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is FokI. This particular enzyme isactive as a dimer. Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA95:10,570-10,575. Accordingly, for the purposes of the presentdisclosure, the portion of the FokI enzyme used in the disclosed fusionproteins is considered a cleavage half-domain. Thus, for targeteddouble-stranded cleavage and/or targeted replacement of cellularsequences using zinc finger-Fold fusions, two fusion proteins, eachcomprising a FokI cleavage half-domain, can be used to reconstitute acatalytically active cleavage domain. Alternatively, a singlepolypeptide molecule containing a zinc finger binding domain and twoFokI cleavage half-domains can also be used. Parameters for targetedcleavage and targeted sequence alteration using zinc finger-Fold fusionsare provided elsewhere in this disclosure.

Exemplary Type IIS restriction enzymes are listed in Table 1. Additionalrestriction enzymes also contain separable binding and cleavage domains,and these are contemplated by the present disclosure. See, for example,Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

TABLE 1 Some Type IIS Restriction Enzymes Aar I BsrB I SspD5 I Ace IIIBsrD I Sth132 I Aci I BstF5 I Sts I Alo I Btr I TspDT I Bae I Bts ITspGW I Bbr7 I Cdi I Tth111 II Bbv I CjeP I UbaP I Bbv II Drd II Bsa IBbvC I Eci I BsmB I Bcc I Eco31 I Bce83 I Eco57 I BceA I Eco57M I Beef IEsp3 I Bcg I Fau I BciV I Fin I Bfi I Fok I Bin I Gdi II Bmg I Gsu IBpu10 I Hga I BsaX I Hin4 II Bsb I Hph I BscA I Ksp632 I BscG I Mbo IIBseR I Mly I BseY I Mme I Bsi I Mnl I Bsm I Pfl1108 I BsmA I Ple I BsmFI Ppi I Bsp24 I Psr I BspG I RleA I BspM I Sap I BspNC I SfaN I Bsr ISim I

Zinc Finger Domain-Cleavage Domain Fusions

Methods for design and construction of fusion proteins (andpolynucleotides encoding same) are known to those of skill in the art.For example, methods for the design and construction of fusion proteincomprising zinc finger proteins (and polynucleotides encoding same) aredescribed in co-owned U.S. Pat. Nos. 6,453,242 and 6,534,261. In certainembodiments, polynucleotides encoding such fusion proteins areconstructed. These polynucleotides can be inserted into a vector and thevector can be introduced into a cell (see below for additionaldisclosure regarding vectors and methods for introducing polynucleotidesinto cells).

In certain embodiments of the methods described herein, a fusion proteincomprises a zinc finger binding domain and a cleavage half-domain fromthe FokI restriction enzyme, and two such fusion proteins are expressedin a cell. Expression of two fusion proteins in a cell can result fromdelivery of the two proteins to the cell; delivery of one protein andone nucleic acid encoding one of the proteins to the cell; delivery oftwo nucleic acids, each encoding one of the proteins, to the cell; or bydelivery of a single nucleic acid, encoding both proteins, to the cell.In additional embodiments, a fusion protein comprises a singlepolypeptide chain comprising two cleavage half domains and a zinc fingerbinding domain. In this case, a single fusion protein is expressed in acell and, without wishing to be bound by theory, is believed to cleaveDNA as a result of formation of an intramolecular dimer of the cleavagehalf-domains.

In general, the components of the fusion proteins (e.g, ZFP-FokIfusions) are arranged such that the zinc finger domain is nearest theamino terminus of the fusion protein, and the cleavage half-domain isnearest the carboxy-terminus. This mirrors the relative orientation ofthe cleavage domain in naturally-occurring dimerizing cleavage domainssuch as those derived from the FokI enzyme, in which the DNA-bindingdomain is nearest the amino terminus and the cleavage half-domain isnearest the carboxy terminus.

In the disclosed fusion proteins, the amino acid sequence between thezinc finger binding domain (which is delimited by the N-terminal most ofthe two conserved cysteine residues and the C-terminal-most of the twoconserved histidine residues) and the cleavage domain (or half-domain)is denoted the “ZC linker.” The ZC linker is to be distinguished fromthe inter-finger linkers discussed above. For instance, in a ZFP-FokIfusion protein (in which the components are arranged: N terminus-zincfinger binding domain-FokI cleavage half domain-C terminus), the ZClinker is located between the second histidine residue of theC-terminal-most zinc finger and the N-terminal-most amino acid residueof the cleavage half-domain (which is generally glutamine (Q) in thesequence QLV). The ZC linker can be any amino acid sequence. To obtainoptimal cleavage, the length of the linker and the distance between thetarget sites (binding sites) are interrelated. See, for example, Smithet al. (2000) Nucleic Acids Res. 28:3361-3369; Bibikova et al. (2001)Mol. Cell. Biol. 21:289-297, noting that their notation for linkerlength differs from that given here. For example, for ZFP-FokI fusionshaving a ZC linker length of four amino acids (as defined herein),optimal cleavage occurs when the binding sites for the fusion proteinsare located 6 or 16 nucleotides apart (as measured from the near edge ofeach binding site).

Methods for Targeted Cleavage

The disclosed methods and compositions can be used to cleave DNA at aregion of interest in cellular chromatin (e.g., at a desired orpredetermined site in a genome, for example, in a gene, either mutant orwild-type). For such targeted DNA cleavage, a zinc finger binding domainis engineered to bind a target site at or near the predeterminedcleavage site, and a fusion protein comprising the engineered zincfinger binding domain and a cleavage domain is expressed in a cell. Uponbinding of the zinc finger portion of the fusion protein to the targetsite, the DNA is cleaved near the target site by the cleavage domain.The exact site of cleavage can depend on the length of the ZC linker.

Alternatively, two fusion proteins, each comprising a zinc fingerbinding domain and a cleavage half-domain, are expressed in a cell, andbind to target sites which are juxtaposed in such a way that afunctional cleavage domain is reconstituted and DNA is cleaved in thevicinity of the target sites. In one embodiment, cleavage occurs betweenthe target sites of the two zinc finger binding domains. One or both ofthe zinc finger binding domains can be engineered.

For targeted cleavage using a zinc finger binding domain-cleavage domainfusion polypeptide, the binding site can encompass the cleavage site, orthe near edge of the binding site can be 1, 2, 3, 4, 5, 6, 10, 25, 50 ormore nucleotides (or any integral value between 1 and 50 nucleotides)from the cleavage site. The exact location of the binding site, withrespect to the cleavage site, will depend upon the particular cleavagedomain, and the length of the ZC linker. For methods in which two fusionpolypeptides, each comprising a zinc finger binding domain and acleavage half-domain, are used, the binding sites generally straddle thecleavage site. Thus the near edge of the first binding site can be 1, 2,3, 4, 5, 6, 10, 25 or more nucleotides (or any integral value between 1and 50 nucleotides) on one side of the cleavage site, and the near edgeof the second binding site can be 1, 2, 3, 4, 5, 6, 10, 25 or morenucleotides (or any integral value between 1 and 50 nucleotides) on theother side of the cleavage site. Methods for mapping cleavage sites invitro and in vivo are known to those of skill in the art.

Thus, the methods described herein can employ an engineered zinc fingerbinding domain fused to a cleavage domain. In these cases, the bindingdomain is engineered to bind to a target sequence, at or near whichcleavage is desired. The fusion protein, or a polynucleotide encodingsame, is introduced into a cell. Once introduced into, or expressed in,the cell, the fusion protein binds to the target sequence and cleaves ator near the target sequence. The exact site of cleavage depends on thenature of the cleavage domain and/or the presence and/or nature oflinker sequences between the binding and cleavage domains. In caseswhere two fusion proteins, each comprising a cleavage half-domain, areused, the distance between the near edges of the binding sites can be 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 25 or more nucleotides (or any integralvalue between 1 and 50 nucleotides). Optimal levels of cleavage can alsodepend on both the distance between the binding sites of the two fusionproteins (See, for example, Smith et al. (2000) Nucleic Acids Res.28:3361-3369; Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297) andthe length of the ZC linker in each fusion protein.

For ZFP-FokI fusion nucleases, the length of the linker between the ZFPand the FokI cleavage half-domain (i.e., the ZC linker) can influencecleavage efficiency. In one experimental system utilizing a ZFP-FokIfusion with a ZC linker of 4 amino acid residues, optimal cleavage wasobtained when the near edges of the binding sites for two ZFP-FokInucleases were separated by 6 base pairs. This particular fusionnuclease comprised the following amino acid sequence between the zincfinger portion and the nuclease half-domain:

(SEQ ID NO: 1) HQRTHQNKKQLVin which the two conserved histidines in the C-terminal portion of thezinc finger and the first three residues in the FokI cleavagehalf-domain are underlined. Accordingly, the linker sequence in thisconstruct is QNKK. Bibikova et al. (2001) Mol. Cell. Biol. 21:289-297.The present inventors have constructed a number of ZFP-FokI fusionnucleases having a variety of ZC linker lengths and sequences, andanalyzed the cleavage efficiencies of these nucleases on a series ofsubstrates having different distances between the ZFP binding sites. SeeExample 4.

In certain embodiments, the cleavage domain comprises two cleavagehalf-domains, both of which are part of a single polypeptide comprisinga binding domain, a first cleavage half-domain and a second cleavagehalf-domain. The cleavage half-domains can have the same amino acidsequence or different amino acid sequences, so long as they function tocleave the DNA.

Cleavage half-domains may also be provided in separate molecules. Forexample, two fusion polypeptides may be introduced into a cell, whereineach polypeptide comprises a binding domain and a cleavage half-domain.The cleavage half-domains can have the same amino acid sequence ordifferent amino acid sequences, so long as they function to cleave theDNA. Further, the binding domains bind to target sequences which aretypically disposed in such a way that, upon binding of the fusionpolypeptides, the two cleavage half-domains are presented in a spatialorientation to each other that allows reconstitution of a cleavagedomain (e.g., by dimerization of the half-domains), thereby positioningthe half-domains relative to each other to form a functional cleavagedomain, resulting in cleavage of cellular chromatin in a region ofinterest. Generally, cleavage by the reconstituted cleavage domainoccurs at a site located between the two target sequences. One or bothof the proteins can be engineered to bind to its target site.

The two fusion proteins can bind in the region of interest in the sameor opposite polarity, and their binding sites (i.e., target sites) canbe separated by any number of nucleotides, e.g., from 0 to 200nucleotides or any integral value therebetween. In certain embodiments,the binding sites for two fusion proteins, each comprising a zinc fingerbinding domain and a cleavage half-domain, can be located between 5 and18 nucleotides apart, for example, 5-8 nucleotides apart, or 15-18nucleotides apart, or 6 nucleotides apart, or 16 nucleotides apart, asmeasured from the edge of each binding site nearest the other bindingsite, and cleavage occurs between the binding sites.

The site at which the DNA is cleaved generally lies between the bindingsites for the two fusion proteins. Double-strand breakage of DNA oftenresults from two single-strand breaks, or “nicks,” offset by 1, 2, 3, 4,5, 6 or more nucleotides, (for example, cleavage of double-stranded DNAby native FokI results from single-strand breaks offset by 4nucleotides). Thus, cleavage does not necessarily occur at exactlyopposite sites on each DNA strand. In addition, the structure of thefusion proteins and the distance between the target sites can influencewhether cleavage occurs adjacent a single nucleotide pair, or whethercleavage occurs at several sites. However, for many applications,including targeted recombination (see infra) cleavage within a range ofnucleotides is generally sufficient, and cleavage between particularbase pairs is not required.

As noted above, the fusion protein(s) can be introduced as polypeptidesand/or polynucleotides. For example, two polynucleotides, eachcomprising sequences encoding one of the aforementioned polypeptides,can be introduced into a cell, and when the polypeptides are expressedand each binds to its target sequence, cleavage occurs at or near thetarget sequence. Alternatively, a single polynucleotide comprisingsequences encoding both fusion polypeptides is introduced into a cell.Polynucleotides can be DNA, RNA or any modified forms or analogues orDNA and/or RNA.

To enhance cleavage specificity, additional compositions may also beemployed in the methods described herein. For example, single cleavagehalf-domains can exhibit limited double-stranded cleavage activity. Inmethods in which two fusion proteins, each containing a three-fingerzinc finger domain and a cleavage half-domain, are introduced into thecell, either protein specifies an approximately 9-nucleotide targetsite. Although the aggregate target sequence of 18 nucleotides is likelyto be unique in a mammalian genome, any given 9-nucleotide target siteoccurs, on average, approximately 23,000 times in the human genome.Thus, non-specific cleavage, due to the site-specific binding of asingle half-domain, may occur. Accordingly, the methods described hereincontemplate the use of a dominant-negative mutant of a cleavagehalf-domain such as FokI (or a nucleic acid encoding same) that isexpressed in a cell along with the two fusion proteins. Thedominant-negative mutant is capable of dimerizing but is unable tocleave, and also blocks the cleavage activity of a half-domain to whichit is dimerized. By providing the dominant-negative mutant in molarexcess to the fusion proteins, only regions in which both fusionproteins are bound will have a high enough local concentration offunctional cleavage half-domains for dimerization and cleavage to occur.At sites where only one of the two fusion proteins are bound, itscleavage half-domain forms a dimer with the dominant negative mutanthalf-domain, and undesirable, non-specific cleavage does not occur.

Three catalytic amino acid residues in the FokI cleavage half-domainhave been identified: Asp 450, Asp 467 and Lys 469. Bitinaite et al.(1998) Proc. Natl. Acad. Sci. USA 95: 10,570-10,575. Thus, one or moremutations at one of these residues can be used to generate a dominantnegative mutation. Further, many of the catalytic amino acid residues ofother Type IIS endonucleases are known and/or can be determined, forexample, by alignment with FokI sequences and/or by generation andtesting of mutants for catalytic activity.

Dimerization Domain Mutations in the Cleavage Half-Domain

Methods for targeted cleavage which involve the use of fusions between aZFP and a cleavage half-domain (such as, e.g., a ZFP/FokI fusion)require the use of two such fusion molecules, each generally directed toa distinct target sequence. Target sequences for the two fusion proteinscan be chosen so that targeted cleavage is directed to a unique site ina genome, as discussed above. A potential source of reduced cleavagespecificity could result from homodimerization of one of the twoZFP/cleavage half-domain fusions. This might occur, for example, due tothe presence, in a genome, of inverted repeats of the target sequencesfor one of the two ZFP/cleavage half-domain fusions, located so as toallow two copies of the same fusion protein to bind with an orientationand spacing that allows formation of a functional dimer.

One approach for reducing the probability of this type of aberrantcleavage at sequences other than the intended target site involvesgenerating variants of the cleavage half-domain that minimize or preventhomodimerization. Preferably, one or more amino acids in the region ofthe half-domain involved in its dimerization are altered. In the crystalstructure of the FokI protein dimer, the structure of the cleavagehalf-domains is reported to be similar to the arrangement of thecleavage half-domains during cleavage of DNA by FokI. Wah et al. (1998)Proc. Natl. Acad. Sci. USA 95:10564-10569. This structure indicates thatamino acid residues at positions 483 and 487 play a key role in thedimerization of the FokI cleavage half-domains. The structure alsoindicates that amino acid residues at positions 446, 447, 479, 483, 484,486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 are allclose enough to the dimerization interface to influence dimerization.Accordingly, amino acid sequence alterations at one or more of theaforementioned positions will likely alter the dimerization propertiesof the cleavage half-domain. Such changes can be introduced, forexample, by constructing a library containing (or encoding) differentamino acid residues at these positions and selecting variants with thedesired properties, or by rationally designing individual mutants. Inaddition to preventing homodimerization, it is also possible that someof these mutations may increase the cleavage efficiency above thatobtained with two wild-type cleavage half-domains.

Accordingly, alteration of a FokI cleavage half-domain at any amino acidresidue which affects dimerization can be used to prevent one of a pairof ZFP/FokI fusions from undergoing homodimerization which can lead tocleavage at undesired sequences. Thus, for targeted cleavage using apair of ZFP FokI fusions, one or both of the fusion proteins cancomprise one or more amino acid alterations that inhibitself-dimerization, but allow heterodimerization of the two fusionproteins to occur such that cleavage occurs at the desired target site.In certain embodiments, alterations are present in both fusion proteins,and the alterations have additive effects; i.e., homodimerization ofeither fusion, leading to aberrant cleavage, is minimized or abolished,while heterodimerization of the two fusion proteins is facilitatedcompared to that obtained with wild-type cleavage half-domains. SeeExample 5.

Methods for Targeted Alteration of Genomic Sequences and TargetedRecombination

Also described herein are methods of replacing a genomic sequence (e.g.,a region of interest in cellular chromatin) with a homologousnon-identical sequence (i.e., targeted recombination). Previous attemptsto replace particular sequences have involved contacting a cell with apolynucleotide comprising sequences bearing homology to a chromosomalregion (i.e., a donor DNA), followed by selection of cells in which thedonor DNA molecule had undergone homologous recombination into thegenome. The success rate of these methods is low, due to poor efficiencyof homologous recombination and a high frequency of non-specificinsertion of the donor DNA into regions of the genome other than thetarget site.

The present disclosure provides methods of targeted sequence alterationcharacterized by a greater efficiency of targeted recombination and alower frequency of non-specific insertion events. The methods involvemaking and using engineered zinc finger binding domains fused tocleavage domains (or cleavage half-domains) to make one or more targeteddouble-stranded breaks in cellular DNA. Because double-stranded breaksin cellular DNA stimulate homologous recombination several thousand-foldin the vicinity of the cleavage site, such targeted cleavage allows forthe alteration or replacement (via homologous recombination) ofsequences at virtually any site in the genome.

In addition to the fusion molecules described herein, targetedreplacement of a selected genomic sequence also requires theintroduction of the replacement (or donor) sequence. The donor sequencecan be introduced into the cell prior to, concurrently with, orsubsequent to, expression of the fusion protein(s). The donorpolynucleotide contains sufficient homology to a genomic sequence tosupport homologous recombination between it and the genomic sequence towhich it bears homology. Approximately 25, 50 100 or 200 nucleotides ormore of sequence homology between a donor and a genomic sequence (or anyintegral value between 10 and 200 nucleotides, or more) will supporthomologous recombination therebetween. Donor sequences can range inlength from 10 to 5,000 nucleotides (or any integral value ofnucleotides therebetween) or longer. It will be readily apparent thatthe donor sequence is typically not identical to the genomic sequencethat it replaces. For example, the sequence of the donor polynucleotidecan contain one or more single base changes, insertions, deletions,inversions or rearrangements with respect to the genomic sequence, solong as sufficient homology is present to support homologousrecombination. Alternatively, a donor sequence can contain anon-homologous sequence flanked by two regions of homology.Additionally, donor sequences can comprise a vector molecule containingsequences that are not homologous to the region of interest in cellularchromatin. Generally, the homologous region(s) of a donor sequence willhave at least 50% sequence identity to a genomic sequence with whichrecombination is desired. In certain embodiments, 60%, 70%, 80%, 90%,95%, 98%, 99%, or 99.9% sequence identity is present. Any value between1% and 100% sequence identity can be present, depending upon the lengthof the donor polynucleotide.

A donor molecule can contain several, discontinuous regions of homologyto cellular chromatin. For example, for targeted insertion of sequencesnot normally present in a region of interest, said sequences can bepresent in a donor nucleic acid molecule and flanked by regions ofhomology to sequence in the region of interest.

To simplify assays (e.g., hybridization, PCR, restriction enzymedigestion) for determining successful insertion of the donor sequence,certain sequence differences may be present in the donor sequence ascompared to the genomic sequence. Preferably, if located in a codingregion, such nucleotide sequence differences will not change the aminoacid sequence, or will make silent amino acid changes (i.e., changeswhich do not affect the structure or function of the protein). The donorpolynucleotide can optionally contain changes in sequences correspondingto the zinc finger domain binding sites in the region of interest, toprevent cleavage of donor sequences that have been introduced intocellular chromatin by homologous recombination.

The donor polynucleotide can be DNA or RNA, single-stranded ordouble-stranded and can be introduced into a cell in linear or circularform. If introduced in linear form, the ends of the donor sequence canbe protected (e.g., from exonucleolytic degradation) by methods known tothose of skill in the art. For example, one or more dideoxynucleotideresidues are added to the 3′ terminus of a linear molecule and/orself-complementary oligonucleotides are ligated to one or both ends.See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA84:4959-4963; Nehls et al. (1996) Science 272:886-889. Additionalmethods for protecting exogenous polynucleotides from degradationinclude, but are not limited to, addition of terminal amino group(s) andthe use of modified internucleotide linkages such as, for example,phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyriboseresidues. A polynucleotide can be introduced into a cell as part of avector molecule having additional sequences such as, for example,replication origins, promoters and genes encoding antibiotic resistance.Moreover, donor polynucleotides can be introduced as naked nucleic acid,as nucleic acid complexed with an agent such as a liposome or poloxamer,or can be delivered by viruses (e.g., adenovirus, AAV).

Without being bound by one theory, it appears that the presence of adouble-stranded break in a cellular sequence, coupled with the presenceof an exogenous DNA molecule having homology to a region adjacent to orsurrounding the break, activates cellular mechanisms which repair thebreak by transfer of sequence information from the donor molecule intothe cellular (e.g., genomic or chromosomal) sequence; i.e., by aprocesses of homologous recombination. Applicants' methodsadvantageously combine the powerful targeting capabilities of engineeredZFPs with a cleavage domain (or cleavage half-domain) to specificallytarget a double-stranded break to the region of the genome at whichrecombination is desired.

For alteration of a chromosomal sequence, it is not necessary for theentire sequence of the donor to be copied into the chromosome, as longas enough of the donor sequence is copied to effect the desired sequencealteration.

The efficiency of insertion of donor sequences by homologousrecombination is inversely related to the distance, in the cellular DNA,between the double-stranded break and the site at which recombination isdesired. In other words, higher homologous recombination efficienciesare observed when the double-stranded break is closer to the site atwhich recombination is desired. In cases in which a precise site ofrecombination is not predetermined (e.g., the desired recombinationevent can occur over an interval of genomic sequence), the length andsequence of the donor nucleic acid, together with the site(s) ofcleavage, are selected to obtain the desired recombination event. Incases in which the desired event is designed to change the sequence of asingle nucleotide pair in a genomic sequence, cellular chromatin iscleaved within 10,000 nucleotides on either side of that nucleotidepair. In certain embodiments, cleavage occurs within 500, 200, 100, 90,80, 70, 60, 50, 40, 30, 20, 10, 5, or 2 nucleotides, or any integralvalue between 2 and 1,000 nucleotides, on either side of the nucleotidepair whose sequence is to be changed.

As detailed above, the binding sites for two fusion proteins, eachcomprising a zinc finger binding domain and a cleavage half-domain, canbe located 5-8 or 15-18 nucleotides apart, as measured from the edge ofeach binding site nearest the other binding site, and cleavage occursbetween the binding sites. Whether cleavage occurs at a single site orat multiple sites between the binding sites is immaterial, since thecleaved genomic sequences are replaced by the donor sequences. Thus, forefficient alteration of the sequence of a single nucleotide pair bytargeted recombination, the midpoint of the region between the bindingsites is within 10,000 nucleotides of that nucleotide pair, preferablywithin 1,000 nucleotides, or 500 nucleotides, or 200 nucleotides, or 100nucleotides, or 50 nucleotides, or 20 nucleotides, or 10 nucleotides, or5 nucleotide, or 2 nucleotides, or one nucleotide, or at the nucleotidepair of interest.

In certain embodiments, a homologous chromosome can serve as the donorpolynucleotide. Thus, for example, correction of a mutation in aheterozygote can be achieved by engineering fusion proteins which bindto and cleave the mutant sequence on one chromosome, but do not cleavethe wild-type sequence on the homologous chromosome. The double-strandedbreak on the mutation-bearing chromosome stimulates a homology-based“gene conversion” process in which the wild-type sequence from thehomologous chromosome is copied into the cleaved chromosome, thusrestoring two copies of the wild-type sequence.

Methods and compositions are also provided that may enhance levels oftargeted recombination including, but not limited to, the use ofadditional ZFP-functional domain fusions to activate expression of genesinvolved in homologous recombination, such as, for example, members ofthe RAD52 epistasis group (e.g., Rad50, Rad51, Rad51B, Rad51C, Rad51D,Rad52, Rad54, Rad54B, Mre11, XRCC2, XRCC3), genes whose productsinteract with the aforementioned gene products (e.g., BRCA1, BRCA2)and/or genes in the NBS1 complex. Similarly ZFP-functional domainfusions can be used, in combination with the methods and compositionsdisclosed herein, to repress expression of genes involved innon-homologous end joining (e.g., Ku70/80, XRCC4, poly(ADP ribose)polymerase, DNA ligase 4). See, for example, Yanez et al. (1998) GeneTherapy 5:149-159; Hoeijmakers (2001) Nature 411:366-374; Johnson et al.(2001) Biochem. Soc. Trans. 29:196-201; Tauchi et al. (2002) Oncogene21:8967-8980. Methods for activation and repression of gene expressionusing fusions between a zinc finger binding domain and a functionaldomain are disclosed in co-owned U.S. Pat. No. 6,534,261. Additionalrepression methods include the use of antisense oligonucleotides and/orsmall interfering RNA (siRNA or RNAi) targeted to the sequence of thegene to be repressed.

As an alternative to or, in addition to, activating expression of geneproducts involved in homologous recombination, fusions of these protein(or functional fragments thereof) with a zinc finger binding domaintargeted to the region of interest, can be used to recruit theseproteins (recombination proteins) to the region of interest, therebyincreasing their local concentration and further stimulating homologousrecombination processes. Alternatively, a polypeptide involved inhomologous recombination as described above (or a functional fragmentthereof) can be part of a triple fusion protein comprising a zinc fingerbinding domain, a cleavage domain (or cleavage half-domain) and therecombination protein (or functional fragment thereof). Additionalproteins involved in gene conversion and recombination-related chromatinremodeling, which can be used in the aforementioned methods andcompositions, include histone acetyltransferases (e.g., Esalp, Tip60),histone methyltransferases (e.g., Dotlp), histone kinases and histonephosphatases.

The p53 protein has been reported to play a central role in repressinghomologous recombination (HR). See, for example, Valerie et al., (2003)Oncogene 22:5792-5812; Janz et al. (2002) Oncogene 21:5929-5933. Forexample, the rate of HR in p53-deficient human tumor lines is10,000-fold greater than in primary human fibroblasts, and there is a100-fold increase in HR in tumor cells with a non-functional p53compared to those with functional p53. Mekeel et al. (1997) Oncogene14:1847-1857. In addition, overexpression of p53 dominant negativemutants leads to a 20-fold increase in spontaneous recombination.Bertrand et al. (1997) Oncogene 14:1117-1122. Analysis of different p53mutations has revealed that the roles of p53 in transcriptionaltransactivation and G1 cell cycle checkpoint control are separable fromits involvement in HR. Saintigny et al. (1999) Oncogene 18:3553-3563;Boehden et al. (2003) Oncogene 22:4111-4117. Accordingly, downregulationof p53 activity can serve to increase the efficiency of targetedhomologous recombination using the methods and compositions disclosedherein. Any method for downregulation of p53 activity can be used,including but not limited to cotransfection and overexpression of a p53dominant negative mutant or targeted repression of p53 gene expressionaccording to methods disclosed, e.g., in co-owned U.S. Pat. No.6,534,261.

Further increases in efficiency of targeted recombination, in cellscomprising a zinc finger/nuclease fusion molecule and a donor DNAmolecule, are achieved by blocking the cells in the G₂ phase of the cellcycle, when homology-driven repair processes are maximally active. Sucharrest can be achieved in a number of ways. For example, cells can betreated with e.g., drugs, compounds and/or small molecules whichinfluence cell-cycle progression so as to arrest cells in G₂ phase.Exemplary molecules of this type include, but are not limited to,compounds which affect microtubule polymerization (e.g., vinblastine,nocodazole, Taxol), compounds that interact with DNA (e.g.,cis-platinum(II) diamine dichloride, Cisplatin, doxorubicin) and/orcompounds that affect DNA synthesis (e.g., thymidine, hydroxyurea,L-mimosine, etoposide, 5-fluorouracil). Additional increases inrecombination efficiency are achieved by the use of histone deacetylase(HDAC) inhibitors (e.g., sodium butyrate, trichostatin A) which alterchromatin structure to make genomic DNA more accessible to the cellularrecombination machinery.

Additional methods for cell-cycle arrest include overexpression ofproteins which inhibit the activity of the CDK cell-cycle kinases, forexample, by introducing a cDNA encoding the protein into the cell or byintroducing into the cell an engineered ZFP which activates expressionof the gene encoding the protein. Cell-cycle arrest is also achieved byinhibiting the activity of cyclins and CDKs, for example, using RNAimethods (e.g., U.S. Pat. No. 6,506,559) or by introducing into the cellan engineered ZFP which represses expression of one or more genesinvolved in cell-cycle progression such as, for example, cyclin and/orCDK genes. See, e.g., co-owned U.S. Pat. No. 6,534,261 for methods forthe synthesis of engineered zinc finger proteins for regulation of geneexpression.

Alternatively, in certain cases, targeted cleavage is conducted in theabsence of a donor polynucleotide (preferably in S or G₂ phase), andrecombination occurs between homologous chromosomes.

Methods to Screen for Cellular Factors that Facilitate HomologousRecombination

Since homologous recombination is a multi-step process requiring themodification of DNA ends and the recruitment of several cellular factorsinto a protein complex, the addition of one or more exogenous factors,along with donor DNA and vectors encoding zinc finger-cleavage domainfusions, can be used to facilitate targeted homologous recombination. Anexemplary method for identifying such a factor or factors employsanalyses of gene expression using microarrays (e.g., Affymetrix GeneChip® arrays) to compare the mRNA expression patterns of differentcells. For example, cells that exhibit a higher capacity to stimulatedouble strand break-driven homologous recombination in the presence ofdonor DNA and zinc finger-cleavage domain fusions, either unaided orunder conditions known to increase the level of gene correction, can beanalyzed for their gene expression patterns compared to cells that lacksuch capacity. Genes that are upregulated or downregulated in a mannerthat directly correlates with increased levels of homologousrecombination are thereby identified and can be cloned into any one of anumber of expression vectors. These expression constructs can beco-transfected along with zinc finger-cleavage domain fusions and donorconstructs to yield improved methods for achieving high-efficiencyhomologous recombination. Alternatively, expression of such genes can beappropriately regulated using engineered zinc finger proteins whichmodulate expression (either activation or repression) of one or morethese genes. See, e.g., co-owned U.S. Pat. No. 6,534,261 for methods forthe synthesis of engineered zinc finger proteins for regulation of geneexpression.

As an example, it was observed that the different clones obtained in theexperiments described in Example 9 and FIG. 27 exhibited a wide-range ofhomologous recombination frequencies, when transfected with donor DNAand plasmids encoding zinc finger-cleavage domain fusions. Geneexpression in clones showing a high frequency of targeted recombinationcan thus be compared to that in clones exhibiting a low frequency, andexpression patterns unique to the former clones can be identified.

As an additional example, studies using cell cycle inhibitors (e.g.,nocodazole or vinblastine, see e.g., Examples 11, 14 and 15) showed thatcells arrested in the G2 phase of the cell cycle carried out homologousrecombination at higher rates, indicating that cellular factorsresponsible for homologous recombination may be preferentially expressedor active in G2. One way to identify these factors is to compare themRNA expression patterns between the stably transfected HEK 293 cellclones that carry out gene correction at high and low levels (e.g.,clone T18 vs. clone T7). Similar comparisons are made between these celllines in response to compounds that arrest the cells in G2 phase.Candidate genes that are differentially expressed in cells that carryout homologous recombination at a higher rate, either unaided or inresponse to compounds that arrest the cells in G2, are identified,cloned, and re-introduced into cells to determine whether theirexpression is sufficient to re-capitulate the improved rates.Alternatively, expression of said candidate genes is activated usingengineered zinc finger transcription factors as described, for example,in co-owned U.S. Pat. No. 6,534,261.

Expression Vectors

A nucleic acid encoding one or more ZFPs or ZFP fusion proteins can becloned into a vector for transformation into prokaryotic or eukaryoticcells for replication and/or expression. Vectors can be prokaryoticvectors, e.g., plasmids, or shuttle vectors, insect vectors, oreukaryotic vectors. A nucleic acid encoding a ZFP can also be clonedinto an expression vector, for administration to a plant cell, animalcell, preferably a mammalian cell or a human cell, fungal cell,bacterial cell, or protozoal cell.

To obtain expression of a cloned gene or nucleic acid, sequencesencoding a ZFP or ZFP fusion protein are typically subcloned into anexpression vector that contains a promoter to direct transcription.Suitable bacterial and eukaryotic promoters are well known in the artand described, e.g., in Sambrook et al., Molecular Cloning, A LaboratoryManual (2nd ed. 1989; 3^(rd) ed., 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., supra. Bacterial expression systemsfor expressing the ZFP are available in, e.g., E. coli, Bacillus sp.,and Salmonella (Palva et al. (1983) Gene 22:229-235). Kits for suchexpression systems are commercially available. Eukaryotic expressionsystems for mammalian cells, yeast, and insect cells are well known bythose of skill in the art and are also commercially available.

The promoter used to direct expression of a ZFP-encoding nucleic aciddepends on the particular application. For example, a strongconstitutive promoter is typically used for expression and purificationof ZFP. In contrast, when a ZFP is administered in vivo for generegulation, either a constitutive or an inducible promoter is used,depending on the particular use of the ZFP. In addition, a preferredpromoter for administration of a ZFP can be a weak promoter, such as HSVTK or a promoter having similar activity. The promoter typically canalso include elements that are responsive to transactivation, e.g.,hypoxia response elements, Gal4 response elements, lac repressorresponse element, and small molecule control systems such astet-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard(1992) PNAS 89:5547; Oligino et al. (1998) Gene Ther. 5:491-496; Wang etal. (1997) Gene Ther. 4:432-441; Neering et al. (1996) Blood88:1147-1155; and Rendahl et al. (1998) Nat. Biotechnol. 16:757-761).The MNDU3 promoter can also be used, and is preferentially active inCD34⁺ hematopoietic stem cells.

In addition to the promoter, the expression vector typically contains atranscription unit or expression cassette that contains all theadditional elements required for the expression of the nucleic acid inhost cells, either prokaryotic or eukaryotic. A typical expressioncassette thus contains a promoter operably linked, e.g., to a nucleicacid sequence encoding the ZFP, and signals required, e.g., forefficient polyadenylation of the transcript, transcriptionaltermination, ribosome binding sites, or translation termination.Additional elements of the cassette may include, e.g., enhancers, andheterologous splicing signals.

The particular expression vector used to transport the geneticinformation into the cell is selected with regard to the intended use ofthe ZFP, e.g., expression in plants, animals, bacteria, fungus,protozoa, etc. (see expression vectors described below). Standardbacterial expression vectors include plasmids such as pBR322-basedplasmids, pSKF, pET23D, and commercially available fusion expressionsystems such as GST and LacZ. An exemplary fusion protein is the maltosebinding protein, “MBP.” Such fusion proteins are used for purificationof the ZFP. Epitope tags can also be added to recombinant proteins toprovide convenient methods of isolation, for monitoring expression, andfor monitoring cellular and subcellular localization, e.g., c-myc orFLAG.

Expression vectors containing regulatory elements from eukaryoticviruses are often used in eukaryotic expression vectors, e.g., SV40vectors, papilloma virus vectors, and vectors derived from Epstein-Barrvirus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+,pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowingexpression of proteins under the direction of the SV40 early promoter,SV40 late promoter, metallothionein promoter, murine mammary tumor viruspromoter, Rous sarcoma virus promoter, polyhedrin promoter, or otherpromoters shown effective for expression in eukaryotic cells.

Some expression systems have markers for selection of stably transfectedcell lines such as thymidine kinase, hygromycin B phosphotransferase,and dihydrofolate reductase. High yield expression systems are alsosuitable, such as using a baculovirus vector in insect cells, with a ZFPencoding sequence under the direction of the polyhedrin promoter orother strong baculovirus promoters.

The elements that are typically included in expression vectors alsoinclude a replicon that functions in E. coli, a gene encoding antibioticresistance to permit selection of bacteria that harbor recombinantplasmids, and unique restriction sites in nonessential regions of theplasmid to allow insertion of recombinant sequences.

Standard transfection methods are used to produce bacterial, mammalian,yeast or insect cell lines that express large quantities of protein,which are then purified using standard techniques (see, e.g., Colley etal. (1989) J. Biol. Chem. 264:17619-17622; Guide to ProteinPurification, in Methods in Enzymology, vol. 182 (Deutscher, ed.,1990)). Transformation of eukaryotic and prokaryotic cells are performedaccording to standard techniques (see, e.g., Morrison (1977) J. Bact.132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362(Wu et al., eds, 1983).

Any of the well known procedures for introducing foreign nucleotidesequences into host cells may be used. These include the use of calciumphosphate transfection, polybrene, protoplast fusion, electroporation,ultrasonic methods (e.g., sonoporation), liposomes, microinjection,naked DNA, plasmid vectors, viral vectors, both episomal andintegrative, and any of the other well known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell (see, e.g., Sambrook et al., supra). It isonly necessary that the particular genetic engineering procedure used becapable of successfully introducing at least one gene into the host cellcapable of expressing the protein of choice.

Nucleic Acids Encoding Fusion Proteins and Delivery to Cells

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding engineered ZFPs in cells (e.g.,mammalian cells) and target tissues. Such methods can also be used toadminister nucleic acids encoding ZFPs to cells in vitro. In certainembodiments, nucleic acids encoding ZFPs are administered for in vivo orex vivo gene therapy uses. Non-viral vector delivery systems include DNAplasmids, naked nucleic acid, and nucleic acid complexed with a deliveryvehicle such as a liposome or poloxamer. Viral vector delivery systemsinclude DNA and RNA viruses, which have either episomal or integratedgenomes after delivery to the cell. For a review of gene therapyprocedures, see Anderson, (1992) Science 256:808-813; Nabel & Felgner(1993) TIBTECH 11:211-217; Mitani & Caskey (1993) TIBTECH 11:162-166;Dillon (1993) TIBTECH 11:167-175; Miller (1992) Nature 357:455-460; VanBrunt (1988) Biotechnology 6(10):1149-1154; Vigne (1995) RestorativeNeurology and Neuroscience 8:35-36; Kremer & Perricaudet (1995) BritishMedical Bulletin 51(1):31-44; Haddada et al., in Current Topics inMicrobiology and Immunology Doerfler and Bohm (eds) (1995); and Yu etal. (1994) Gene Therapy 1:13-26.

Methods of non-viral delivery of nucleic acids encoding engineered ZFPsinclude electroporation, lipofection, microinjection, biolistics,virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acidconjugates, naked DNA, artificial virions, and agent-enhanced uptake ofDNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) canalso be used for delivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.) and BTX Molecular Delivery Systems (Holliston, Mass.).

Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787;and 4,897,355) and lipofection reagents are sold commercially (e.g.,Transfectam™ and Lipofectin™). Cationic and neutral lipids that aresuitable for efficient receptor-recognition lipofection ofpolynucleotides include those of Felgner, International PatentPublication Nos. WO 91/17424, WO 91/16024. Delivery can be to cells (exvivo administration) or target tissues (in vivo administration).

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal (1995) Science 270:404-410; Blaese et al.(1995) Cancer Gene Ther. 2:291-297; Behr et al. (1994) BioconjugateChem. 5:382-389; Remy et al. (1994) Bioconjugate Chem. 5:647-654; Gao etal. (1995) Gene Therapy 2:710-722; Ahmad et al. (1992) Cancer Res.52:4817-4820; U.S. Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975;4,485,054; 4,501,728; 4,774,085; 4,837,028; and 4,946,787).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to patients (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to patients (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SIV), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al. (1992) J. Virol.66:2731-2739; Johann et al. (1992) J. Virol. 66:1635-1640; Sommerfelt etal. (1990) Virol. 176:58-59; Wilson et al. (1989) J. Virol.63:2374-2378; Miller et al. (1991) J. Virol. 65:2220-2224; InternationalPatent Publication No. WO 94/26877).

In applications in which transient expression of a ZFP fusion protein ispreferred, adenoviral based systems can be used. Adenoviral basedvectors are capable of very high transduction efficiency in many celltypes and do not require cell division. With such vectors, high titerand high levels of expression have been obtained. This vector can beproduced in large quantities in a relatively simple system.Adeno-associated virus (“AAV”) vectors are also used to transduce cellswith target nucleic acids, e.g., in the in vitro production of nucleicacids and peptides, and for in vivo and ex vivo gene therapy procedures(see, e.g., West et al. (1987) Virology 160:38-47; U.S. Pat. No.4,797,368; International Patent Publication No. WO 93/24641; Kotin(1994) Human Gene Therapy 5:793-801; Muzyczka (1994) J. Clin. Invest.94:1351. Construction of recombinant AAV vectors are described in anumber of publications, including U.S. Pat. No. 5,173,414; Tratschin etal. (1985) Mol. Cell. Biol. 5:3251-3260; Tratschin et al. (1984) Mol.Cell. Biol. 4:2072-2081; Hermonat & Muzyczka (1984) PNAS 81:6466-6470;and Samulski et al. (1989) J. Virol. 63:03822-3828.

Adeno-associated virus vectors include AAV serotypes 1, 2, 5, 6, 7, 8and 9; as well as chimeric AAV serotypes, e.g., AAV 2/1 and AAV 2/5.Both single-stranded and double-stranded (e.g., self-complementary) AAVvectors can be used.

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al. (1995) Blood 85:3048-305; Kohn et al.(1995) Nat. Med. 1:1017-102; Malech et al. (1997) PNAS94(22):12133-12138). PA317/pLASN was the first therapeutic vector usedin a gene therapy trial. (Blaese et al. (1995) Science 270:475-480).Transduction efficiencies of 50% or greater have been observed for MFG-Spackaged vectors. (Ellem et al. (1997) Immunol Immunother 44(1):10-20;Dranoff et al. (1997) Hum. Gene Ther. 1:111-2.

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al. (1998) Lancet 351(9117):1702-3, Kearns et al. (1996) GeneTher. 9:748-55).

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including nondividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for antitumorimmunization with intramuscular injection (Sterman et al. (1998) Hum.Gene Ther. 7:1083-9). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.(1996) Infection 24(1):5-10; Sterman et al. (1998) Hum. Gene Ther.9(7):1083-1089; Welsh et al. (1995) Hum. Gene Ther. 2:205-18; Alvarez etal. (1997) Hum. Gene Ther. 5:597-613; Topf et al. (1998) Gene Ther.5:507-513; Sterman et al. (1998) Hum. Gene Ther. 7:1083-1089.

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and w2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al. (1995) Proc. Natl.Acad. Sci. USA 92:9747-9751, reported that Moloney murine leukemia viruscan be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Ex vivo cell transfection for diagnostics, research, or for gene therapy(e.g., via re-infusion of the transfected cells into the host organism)is well known to those of skill in the art. In a preferred embodiment,cells are isolated from the subject organism, transfected with a ZFPnucleic acid (gene or cDNA), and re-infused back into the subjectorganism (e.g., patient). Various cell types suitable for ex vivotransfection are well known to those of skill in the art (see, e.g.,Freshney et al., Culture of Animal Cells, A Manual of Basic Technique(3rd ed. 1994)) and the references cited therein for a discussion of howto isolate and culture cells from patients).

In one embodiment, stem cells are used in ex vivo procedures for celltransfection and gene therapy. The advantage to using stem cells is thatthey can be differentiated into other cell types in vitro, or can beintroduced into a mammal (such as the donor of the cells) where theywill engraft in the bone marrow. Methods for differentiating CD34+ cellsin vitro into clinically important immune cell types using cytokinessuch a GM-CSF, IFN-γ and TNF-α are known (see Inaba et al. (1992) J.Exp. Med. 176:1693-1702).

Stem cells are isolated for transduction and differentiation using knownmethods. For example, stem cells are isolated from bone marrow cells bypanning the bone marrow cells with antibodies which bind unwanted cells,such as CD4+ and CD8+(T cells), CD45+(panB cells), GR-1 (granulocytes),and Tad (differentiated antigen presenting cells) (see Inaba et al.(1992) J. Exp. Med. 176:1693-1702).

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingtherapeutic ZFP nucleic acids can also be administered directly to anorganism for transduction of cells in vivo. Alternatively, naked DNA canbe administered. Administration is by any of the routes normally usedfor introducing a molecule into ultimate contact with blood or tissuecells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Methods for introduction of DNA into hematopoietic stem cells aredisclosed, for example, in U.S. Pat. No. 5,928,638.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

DNA constructs may be introduced into the genome of a desired plant hostby a variety of conventional techniques. For reviews of such techniquessee, for example, Weissbach & Weissbach Methods for Plant MolecularBiology (1988, Academic Press, N.Y.) Section VIII, pp. 421-463; andGrierson & Corey, Plant Molecular Biology (1988, 2d Ed.), Blackie,London, Ch. 7-9. For example, the DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation and microinjection of plant cell protoplasts, or the DNAconstructs can be introduced directly to plant tissue using biolisticmethods, such as DNA particle bombardment (see, e.g., Klein et al.(1987) Nature 327:70-73). Alternatively, the DNA constructs may becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, for example Horsch et al. (1984) Science 233:496-498, and Fraley etal. (1983) Proc. Nat'l. Acad. Sci. USA 80:4803. The virulence functionsof the Agrobacterium tumefaciens host will direct the insertion of theconstruct and adjacent marker into the plant cell DNA when the cell isinfected by the bacteria using binary T DNA vector (Bevan (1984) Nuc.Acid Res. 12:8711-8721) or the co-cultivation procedure (Horsch et al.(1985) Science 227:1229-1231). Generally, the Agrobacteriumtransformation system is used to engineer dicotyledonous plants (Bevanet al. (1982) Ann. Rev. Genet 16:357-384; Rogers et al. (1986) MethodsEnzymol. 118:627-641). The Agrobacterium transformation system may alsobe used to transform, as well as transfer, DNA to monocotyledonousplants and plant cells. See Hernalsteen et al. (1984) EMBO J3:3039-3041; Hooykass-Van Slogteren et al. (1984) Nature 311:763-764;Grimsley et al. (1987) Nature 325:1677-179; Boulton et al. (1989) PlantMol. Biol. 12:31-40; and Gould et al. (1991) Plant Physiol. 95:426-434.

Alternative gene transfer and transformation methods include, but arenot limited to, protoplast transformation through calcium-, polyethyleneglycol (PEG)- or electroporation-mediated uptake of naked DNA (seePaszkowski et al. (1984) EMBO J 3:2717-2722, Potrykus et al. (1985)Molec. Gen. Genet. 199:169-177; Fromm et al. (1985) Proc. Nat. Acad.Sci. USA 82:5824-5828; and Shimamoto (1989) Nature 338:274-276) andelectroporation of plant tissues (D'Halluin et al. (1992) Plant Cell4:1495-1505). Additional methods for plant cell transformation includemicroinjection, silicon carbide mediated DNA uptake (Kaeppler et al.(1990) Plant Cell Reporter 9:415-418), and microprojectile bombardment(see Klein et al. (1988) Proc. Nat. Acad. Sci. USA 85:4305-4309; andGordon-Kamm et al. (1990) Plant Cell 2:603-618).

The disclosed methods and compositions can be used to insert exogenoussequences into a predetermined location in a plant cell genome. This isuseful inasmuch as expression of an introduced transgene into a plantgenome depends critically on its integration site. Accordingly, genesencoding, e.g., nutrients, antibiotics or therapeutic molecules can beinserted, by targeted recombination, into regions of a plant genomefavorable to their expression.

Transformed plant cells which are produced by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype and thus the desired phenotype.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide and/or herbicide marker which has been introduced together withthe desired nucleotide sequences. Plant regeneration from culturedprotoplasts is described in Evans, et al., “Protoplasts Isolation andCulture” in Handbook of Plant Cell Culture, pp. 124-176, MacmillianPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, pollens,embryos or parts thereof. Such regeneration techniques are describedgenerally in Klee et al. (1987) Ann. Rev. of Plant Phys. 38:467-486.

Nucleic acids introduced into a plant cell can be used to confer desiredtraits on essentially any plant. A wide variety of plants and plant cellsystems may be engineered for the desired physiological and agronomiccharacteristics described herein using the nucleic acid constructs ofthe present disclosure and the various transformation methods mentionedabove. In preferred embodiments, target plants and plant cells forengineering include, but are not limited to, those monocotyledonous anddicotyledonous plants, such as crops including grain crops (e.g., wheat,maize, rice, millet, barley), fruit crops (e.g., tomato, apple, pear,strawberry, orange), forage crops (e.g., alfalfa), root vegetable crops(e.g., carrot, potato, sugar beets, yam), leafy vegetable crops (e.g.,lettuce, spinach); flowering plants (e.g., petunia, rose,chrysanthemum), conifers and pine trees (e.g., pine fir, spruce); plantsused in phytoremediation (e.g., heavy metal accumulating plants); oilcrops (e.g., sunflower, rape seed) and plants used for experimentalpurposes (e.g., Arabidopsis). Thus, the disclosed methods andcompositions have use over a broad range of plants, including, but notlimited to, species from the genera Asparagus, Avena, Brassica, Citrus,Citrullus, Capsicum, Cucurbita, Daucus, Glycine, Hordeum, Lactuca,Lycopersicon, Malus, Manihot, Nicotiana, Oryza, Persea, Pisum, Pyrus,Prunus, Raphanus, Secale, Solanum, Sorghum, Triticum, Vitis, Vigna, andZea.

One of skill in the art will recognize that after the expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

A transformed plant cell, callus, tissue or plant may be identified andisolated by selecting or screening the engineered plant material fortraits encoded by the marker genes present on the transforming DNA. Forinstance, selection may be performed by growing the engineered plantmaterial on media containing an inhibitory amount of the antibiotic orherbicide to which the transforming gene construct confers resistance.Further, transformed plants and plant cells may also be identified byscreening for the activities of any visible marker genes (e.g., theβ-glucuronidase, luciferase, B or C1 genes) that may be present on therecombinant nucleic acid constructs. Such selection and screeningmethodologies are well known to those skilled in the art.

Physical and biochemical methods also may be used to identify plant orplant cell transformants containing inserted gene constructs. Thesemethods include but are not limited to: 1) Southern analysis or PCRamplification for detecting and determining the structure of therecombinant DNA insert; 2) Northern blot, 51 RNase protection,primer-extension or reverse transcriptase-PCR amplification fordetecting and examining RNA transcripts of the gene constructs; 3)enzymatic assays for detecting enzyme or ribozyme activity, where suchgene products are encoded by the gene construct; 4) protein gelelectrophoresis, Western blot techniques, immunoprecipitation, orenzyme-linked immunoassays, where the gene construct products areproteins. Additional techniques, such as in situ hybridization, enzymestaining, and immunostaining, also may be used to detect the presence orexpression of the recombinant construct in specific plant organs andtissues. The methods for doing all these assays are well known to thoseskilled in the art.

Effects of gene manipulation using the methods disclosed herein can beobserved by, for example, northern blots of the RNA (e.g., mRNA)isolated from the tissues of interest. Typically, if the amount of mRNAhas increased, it can be assumed that the corresponding endogenous geneis being expressed at a greater rate than before. Other methods ofmeasuring gene and/or CYP74B activity can be used. Different types ofenzymatic assays can be used, depending on the substrate used and themethod of detecting the increase or decrease of a reaction product orby-product. In addition, the levels of and/or CYP74B protein expressedcan be measured immunochemically, i.e., ELISA, RIA, EIA and otherantibody based assays well known to those of skill in the art, such asby electrophoretic detection assays (either with staining or westernblotting). The transgene may be selectively expressed in some tissues ofthe plant or at some developmental stages, or the transgene may beexpressed in substantially all plant tissues, substantially along itsentire life cycle. However, any combinatorial expression mode is alsoapplicable.

The present disclosure also encompasses seeds of the transgenic plantsdescribed above wherein the seed has the transgene or gene construct.The present disclosure further encompasses the progeny, clones, celllines or cells of the transgenic plants described above wherein saidprogeny, clone, cell line or cell has the transgene or gene construct.

Delivery Vehicles

An important factor in the administration of polypeptide compounds, suchas ZFP fusion proteins, is ensuring that the polypeptide has the abilityto traverse the plasma membrane of a cell, or the membrane of anintra-cellular compartment such as the nucleus. Cellular membranes arecomposed of lipid-protein bilayers that are freely permeable to small,nonionic lipophilic compounds and are inherently impermeable to polarcompounds, macromolecules, and therapeutic or diagnostic agents.However, proteins and other compounds such as liposomes have beendescribed, which have the ability to translocate polypeptides such asZFPs across a cell membrane.

For example, “membrane translocation polypeptides” have amphiphilic orhydrophobic amino acid subsequences that have the ability to act asmembrane-translocating carriers. In one embodiment, homeodomain proteinshave the ability to translocate across cell membranes. The shortestinternalizable peptide of a homeodomain protein, Antennapedia, was foundto be the third helix of the protein, from amino acid position 43 to 58(see, e.g., Prochiantz (1996) Current Opinion in Neurobiology6:629-634). Another subsequence, the h (hydrophobic) domain of signalpeptides, was found to have similar cell membrane translocationcharacteristics (see, e.g., Lin et al. (1995) J. Biol. Chem.270(1):4255-14258).

Examples of peptide sequences which can be linked to a protein, forfacilitating uptake of the protein into cells, include, but are notlimited to: an 11 amino acid peptide of the tat protein of HIV; a 20residue peptide sequence which corresponds to amino acids 84-103 of thep16 protein (see Fahraeus et al. (1996) Current Biology 6:84); the thirdhelix of the 60-amino acid long homeodomain of Antennapedia (Derossi etal. (1994) J. Biol. Chem. 269:10444); the h region of a signal peptidesuch as the Kaposi fibroblast growth factor (K-FGF) h region (Lin etal., supra); or the VP22 translocation domain from HSV (Elliot & O'Hare(1997) Cell 88:223-233). Other suitable chemical moieties that provideenhanced cellular uptake may also be chemically linked to ZFPs. Membranetranslocation domains (i.e., internalization domains) can also beselected from libraries of randomized peptide sequences. See, forexample, Yeh et al. (2003) Molecular Therapy 7(5):S461, Abstract #1191.

Toxin molecules also have the ability to transport polypeptides acrosscell membranes. Often, such molecules (called “binary toxins”) arecomposed of at least two parts: a translocation/binding domain orpolypeptide and a separate toxin domain or polypeptide. Typically, thetranslocation domain or polypeptide binds to a cellular receptor, andthen the toxin is transported into the cell. Several bacterial toxins,including Clostridium perfringens iota toxin, diphtheria toxin (DT),Pseudomonas exotoxin A (PE), pertussis toxin (PT), Bacillus anthracistoxin, and pertussis adenylate cyclase (CYA), have been used to deliverpeptides to the cell cytosol as internal or amino-terminal fusions(Arora et al. (1993) J. Biol. Chem. 268:3334-3341; Perelle et al. (1993)Infect. Immun. 61:5147-5156; Stenmark et al. (1991) J Cell Biol.113:1025-1032; Donnelly et al. (1993) PNAS 90:3530-3534; Carbonetti etal. (1995) Abstr. Annu. Meet. Am. Soc. Microbiol. 95:295; Sebo et al.(1995) Infect. Immun. 63:3851-3857; Klimpel et al. (1992) PNAS U.S.A.89:10277-10281; and Novak et al. (1992) J. Biol. Chem. 267:17186-17193).

Such peptide sequences can be used to translocate ZFPs across a cellmembrane. ZFPs can be conveniently fused to or derivatized with suchsequences. Typically, the translocation sequence is provided as part ofa fusion protein. Optionally, a linker can be used to link the ZFP andthe translocation sequence. Any suitable linker can be used, e.g., apeptide linker.

The ZFP can also be introduced into an animal cell, preferably amammalian cell, via a liposomes and liposome derivatives such asimmunoliposomes. The term “liposome” refers to vesicles comprised of oneor more concentrically ordered lipid bilayers, which encapsulate anaqueous phase. The aqueous phase typically contains the compound to bedelivered to the cell, i.e., a ZFP.

The liposome fuses with the plasma membrane, thereby releasing the druginto the cytosol. Alternatively, the liposome is phagocytosed or takenup by the cell in a transport vesicle. Once in the endosome orphagosome, the liposome either degrades or fuses with the membrane ofthe transport vesicle and releases its contents.

In current methods of drug delivery via liposomes, the liposomeultimately becomes permeable and releases the encapsulated compound (inthis case, a ZFP) at the target tissue or cell. For systemic or tissuespecific delivery, this can be accomplished, for example, in a passivemanner wherein the liposome bilayer degrades over time through theaction of various agents in the body. Alternatively, active drug releaseinvolves using an agent to induce a permeability change in the liposomevesicle. Liposome membranes can be constructed so that they becomedestabilized when the environment becomes acidic near the liposomemembrane (see, e.g., Wang et al. (1987) PNAS 84(22):7851-5; Biochemistry28:908 (1989)). When liposomes are endocytosed by a target cell, forexample, they become destabilized and release their contents. Thisdestabilization is termed fusogenesis. Dioleoylphosphatidylethanolamine(DOPE) is the basis of many “fusogenic” systems.

Such liposomes typically comprise a ZFP and a lipid component, e.g., aneutral and/or cationic lipid, optionally including areceptor-recognition molecule such as an antibody that binds to apredetermined cell surface receptor or ligand (e.g., an antigen). Avariety of methods are available for preparing liposomes as describedin, e.g., Szoka et al. (1980) Ann. Rev. Biophys. Bioeng. 9:467; U.S.Pat. Nos. 4,186,183; 4,217,344; 4,235,871; 4,261,975; 4,485,054;4,501,728; 4,774,085; 4,837,028; 4,235,871; 4,261,975; 4,485,054;4,501,728; 4,774,085; 4,837,028; 4,946,787; International PatentPublication No. WO 91/17424, Deamer & Bangham, Biochim. Biophys. Acta(1976) 443:629-634; Fraley et al. (1979) PNAS 76:3348-3352; Hope et al.(1985) Biochim. Biophys. Acta 812:55-65; Mayer et al. (1986) Biochim.Biophys. Acta 858:161-168; Williams et al. (1988) PNAS 85:242-246;Liposomes (Ostro (ed.), 1983, Chapter 1); Hope et al. (1986) Chem. Phys.Lip. 40:89; Gregoriadis, Liposome Technology (1984) and Lasic,Liposomes: from Physics to Applications (1993)). Suitable methodsinclude, for example, sonication, extrusion, highpressure/homogenization, microfluidization, detergent dialysis,calcium-induced fusion of small liposome vesicles and ether-fusionmethods, all of which are known to those of skill in the art.

In certain embodiments, it is desirable to target liposomes usingtargeting moieties that are specific to a particular cell type, tissue,and the like. Targeting of liposomes using a variety of targetingmoieties (e.g., ligands, receptors, and monoclonal antibodies) has beendescribed. See, e.g., U.S. Pat. Nos. 4,957,773 and 4,603,044.

Examples of targeting moieties include monoclonal antibodies specific toantigens associated with neoplasms, such as prostate cancer specificantigen and MAGE. Tumors can also be diagnosed by detecting geneproducts resulting from the activation or over-expression of oncogenes,such as ras or c-erbB2. In addition, many tumors express antigensnormally expressed by fetal tissue, such as the alphafetoprotein (AFP)and carcinoembryonic antigen (CEA). Sites of viral infection can bediagnosed using various viral antigens such as hepatitis B core andsurface antigens (HBVc, HBVs) hepatitis C antigens, Epstein-Barr virusantigens, human immunodeficiency type-1 virus (HIV1) and papilloma virusantigens. Inflammation can be detected using molecules specificallyrecognized by surface molecules which are expressed at sites ofinflammation such as integrins (e.g., VCAM-1), selectin receptors (e.g.,ELAM-1) and the like.

Standard methods for coupling targeting agents to liposomes can be used.These methods generally involve incorporation into liposomes of lipidcomponents, e.g., phosphatidylethanolamine, which can be activated forattachment of targeting agents, or derivatized lipophilic compounds,such as lipid derivatized bleomycin. Antibody targeted liposomes can beconstructed using, for instance, liposomes which incorporate protein A(see Renneisen et al. (1990) J. Biol. Chem. 265:16337-16342 and Leonettiet al. (1990) PNAS 87:2448-2451.

Dosages

For therapeutic applications, the dose administered to a patient, or toa cell which will be introduced into a patient, in the context of thepresent disclosure, should be sufficient to effect a beneficialtherapeutic response in the patient over time. In addition, particulardosage regimens can be useful for determining phenotypic changes in anexperimental setting, e.g., in functional genomics studies, and in cellor animal models. The dose will be determined by the efficacy and K_(d)of the particular ZFP employed, the nuclear volume of the target cell,and the condition of the patient, as well as the body weight or surfacearea of the patient to be treated. The size of the dose also will bedetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular compoundor vector in a particular patient.

The maximum therapeutically effective dosage of ZFP for approximately99% binding to target sites is calculated to be in the range of lessthan about 1.5×10⁵ to 1.5×10⁶ copies of the specific ZFP molecule percell. The number of ZFPs per cell for this level of binding iscalculated as follows, using the volume of a HeLa cell nucleus(approximately 1000 μm³ or 10⁻¹² L; Cell Biology, (Altman & Katz, eds.(1976)). As the HeLa nucleus is relatively large, this dosage number isrecalculated as needed using the volume of the target cell nucleus. Thiscalculation also does not take into account competition for ZFP bindingby other sites. This calculation also assumes that essentially all ofthe ZFP is localized to the nucleus. A value of 100×K_(d) is used tocalculate approximately 99% binding of to the target site, and a valueof 10×K_(d) is used to calculate approximately 90% binding of to thetarget site. For this example, K_(d)=25 nM

-   -   ZFP+target site↔complex    -   i.e., DNA+protein↔DNA:protein complex

$K_{d} = \frac{\lbrack{DNA}\rbrack \lbrack{protein}\rbrack}{\left\lbrack {{DNA}\text{:}{protein}\mspace{14mu} {complex}} \right\rbrack}$

-   -   -   When 50% of ZFP is bound, K_(d)=[protein]        -   So when [protein]=25 nM and the nucleus volume is 10⁻¹² L        -   [protein]=(25×10⁻⁹ moles/L) (10⁻¹² L/nucleus) (6×10²³            molecules/mole)        -   =15,000 molecules/nucleus for 50% binding        -   When 99% target is bound; 100×K_(d)=[protein]        -   100×K_(d)=[protein]=2.5 μM        -   (2.5×10⁻⁶ moles/L) (10⁻¹² L/nucleus) (6×10²³ molecules/mole)        -   =about 1,500,000 molecules per nucleus for 99% binding of            target site.

The appropriate dose of an expression vector encoding a ZFP can also becalculated by taking into account the average rate of ZFP expressionfrom the promoter and the average rate of ZFP degradation in the cell.In certain embodiments, a weak promoter such as a wild-type or mutantHSV TK promoter is used, as described above. The dose of ZFP inmicrograms is calculated by taking into account the molecular weight ofthe particular ZFP being employed.

In determining the effective amount of the ZFP to be administered in thetreatment or prophylaxis of disease, the physician evaluates circulatingplasma levels of the ZFP or nucleic acid encoding the ZFP, potential ZFPtoxicities, progression of the disease, and the production of anti-ZFPantibodies. Administration can be accomplished via single or divideddoses.

Pharmaceutical Compositions and Administration

ZFPs and expression vectors encoding ZFPs can be administered directlyto the patient for targeted cleavage and/or recombination, and fortherapeutic or prophylactic applications, for example, cancer, ischemia,diabetic retinopathy, macular degeneration, rheumatoid arthritis,psoriasis, HIV infection, sickle cell anemia, Alzheimer's disease,muscular dystrophy, neurodegenerative diseases, vascular disease, cysticfibrosis, stroke, and the like. Examples of microorganisms that can beinhibited by ZFP gene therapy include pathogenic bacteria, e.g.,chlamydia, rickettsial bacteria, mycobacteria, staphylococci,streptococci, pneumococci, meningococci and conococci, klebsiella,proteus, serratia, pseudomonas, legionella, diphtheria, salmonella,bacilli, cholera, tetanus, botulism, anthrax, plague, leptospirosis, andLyme disease bacteria; infectious fungus, e.g., Aspergillus, Candidaspecies; protozoa such as sporozoa (e.g., Plasmodia), rhizopods (e.g.,Entamoeba) and flagellates (Trypanosoma, Leishmania, Trichomonas,Giardia, etc.); viral diseases, e.g., hepatitis (A, B, or C), herpesvirus (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), HIV, Ebola,adenovirus, influenza virus, flaviviruses, echovirus, rhinovirus,coxsackie virus, coronavirus, respiratory syncytial virus, mumps virus,rotavirus, measles virus, rubella virus, parvovirus, vaccinia virus,HTLV virus, dengue virus, papillomavirus, poliovirus, rabies virus, andarboviral encephalitis virus, etc.

Administration of therapeutically effective amounts is by any of theroutes normally used for introducing ZFP into ultimate contact with thetissue to be treated. The ZFPs are administered in any suitable manner,preferably with pharmaceutically acceptable carriers. Suitable methodsof administering such modulators are available and well known to thoseof skill in the art, and, although more than one route can be used toadminister a particular composition, a particular route can oftenprovide a more immediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions that areavailable (see, e.g., Remington's Pharmaceutical Sciences, 17^(th) ed.1985)).

The ZFPs, alone or in combination with other suitable components, can bemade into aerosol formulations (i.e., they can be “nebulized”) to beadministered via inhalation. Aerosol formulations can be placed intopressurized acceptable propellants, such as dichlorodifluoromethane,propane, nitrogen, and the like.

Formulations suitable for parenteral administration, such as, forexample, by intravenous, intramuscular, intradermal, and subcutaneousroutes, include aqueous and non-aqueous, isotonic sterile injectionsolutions, which can contain antioxidants, buffers, bacteriostats, andsolutes that render the formulation isotonic with the blood of theintended recipient, and aqueous and non-aqueous sterile suspensions thatcan include suspending agents, solubilizers, thickening agents,stabilizers, and preservatives. The disclosed compositions can beadministered, for example, by intravenous infusion, orally, topically,intraperitoneally, intravesically or intrathecally. The formulations ofcompounds can be presented in unit-dose or multi-dose sealed containers,such as ampules and vials. Injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described.

Applications

The disclosed methods and compositions for targeted cleavage can be usedto induce mutations in a genomic sequence, e.g., large deletionmutations. Generation of targeted deletions, as disclosed herein, can beused to create gene knock-outs (e.g., for functional genomics or targetvalidation) and for purposes of cell engineering or proteinoverexpression.

Targeted cleavage at a site in chromosomal DNA requires a pair of zincfinger/nuclease half-domain fusion proteins (ZFNs) so that dimerizationof the cleavage half-domains occurs. Accordingly, for targeted deletionof long sequences, two pairs of ZFNs are used, to cleave at two sitesand delete sequences between the two cleavage sites.

Targeted deletion of infecting or integrated viral genomes can be usedto treat viral infections in a host. Additionally, targeted deletion ofgenes encoding receptors for viruses can be used to block expression ofsuch receptors, thereby preventing viral infection and/or viral spreadin a host organism. Targeted deletion of genes encoding viral receptors(e.g., the CCR5 and CXCR4 receptors for HIV) can be used to render thereceptors unable to bind to virus, thereby preventing new infection andblocking the spread of existing infections. Non-limiting examples ofviruses or viral receptors that may be targeted include herpes simplexvirus (HSV), such as HSV-1 and HSV-2, varicella zoster virus (VZV),Epstein-Barr virus (EBV) and cytomegalovirus (CMV), HHV6 and HHV7. Thehepatitis family of viruses includes hepatitis A virus (HAV), hepatitisB virus (HBV), hepatitis C virus (HCV), the delta hepatitis virus (HDV),hepatitis E virus (HEV) and hepatitis G virus (HGV). Other viruses ortheir receptors may be targeted, including, but not limited to,Picornaviridae (e.g., polioviruses, etc.); Caliciviridae; Togaviridae(e.g., rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae;Reoviridae; Birnaviridae; Rhabodoviridae (e.g., rabies virus, etc.);Filoviridae; Paramyxoviridae (e.g., mumps virus, measles virus,respiratory syncytial virus, etc.); Orthomyxoviridae (e.g., influenzavirus types A, B and C, etc.); Bunyaviridae; Arenaviridae; Retroviradae;lentiviruses (e.g., HTLV-I; HTLV-II; HIV-1 (also known as HTLV-III, LAV,ARV, hTLR, etc.) HIV-II); simian immunodeficiency virus (SIV), humanpapillomavirus (HPV), influenza virus and the tick-borne encephalitisviruses. See, e.g. Virology, 3rd Edition (W. K. Joklik ed. 1988);Fundamental Virology, 2nd Edition (B. N. Fields and D. M. Knipe, eds.1991), for a description of these and other viruses. Receptors for HIV,for example, include CCR-5 and CXCR-4.

In similar fashion, the genome of an infecting bacterium can bemutagenized by targeted deletion, to block or ameliorate bacterialinfections.

Certain genetic diseases result from expression of a mutant geneproduct. In such cases, inactivation of the mutant gene product bytargeted deletion of its gene may ameliorate or cure the disease.Exemplary genetic diseases include, but are not limited to,achondroplasia, achromatopsia, acid maltase deficiency, adenosinedeaminase deficiency (OMIM No. 102700), adrenoleukodystrophy, aicardisyndrome, alpha-1 antitrypsin deficiency, alpha-thalassemia, androgeninsensitivity syndrome, apert syndrome, arrhythmogenic rightventricular, dysplasia, ataxia telangictasia, barth syndrome,beta-thalassemia, blue rubber bleb nevus syndrome, canavan disease,chronic granulomatous diseases (CGD), cri du chat syndrome, cysticfibrosis, dercum's disease, ectodermal dysplasia, fanconi anemia,fibrodysplasia ossificans progressive, fragile X syndrome, galactosemis,Gaucher's disease, generalized gangliosidoses (e.g., GM1),hemochromatosis, the hemoglobin C mutation in the 6^(th) codon ofbeta-globin (HbC), hemophilia, Huntington's disease, Hurler Syndrome,hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-GiedionSyndrome, leukocyte adhesion deficiency (LAD, OMIM No. 116920),leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome,mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetesinsipdius, neurofibromatosis, Neimann-Pick disease, osteogenesisimperfecta, porphyria, Prader-Willi syndrome, progeria, Proteussyndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome,Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachmansyndrome, sickle cell disease (sickle cell anemia), Smith-Magenissyndrome, Stickler syndrome, Tay-Sachs disease, Thrombocytopenia AbsentRadius (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberoussclerosis, Turner's syndrome, urea cycle disorder, von Hippel-Landaudisease, Waardenburg syndrome, Williams syndrome, Wilson's disease,Wiskott-Aldrich syndrome, X-linked lymphoproliferative syndrome (XLP,OMIM No. 308240), acquired immunodeficiencies, lysosomal storagediseases (e.g., Gaucher's disease, GM1, Fabry disease and Tay-Sachsdisease), mucopolysaccahidosis (e.g. Hunter's disease, Hurler'sdisease), hemoglobinopathies (e.g., sickle cell diseases, HbC,α-thalassemia, β-thalassemia) and hemophilias.

In certain cases, alteration of a genomic sequence in a pluripotent cell(e.g., a hematopoietic stem cell) is desired. Methods for mobilization,enrichment and culture of hematopoietic stem cells are known in the art.See for example, U.S. Pat. Nos. 5,061,620; 5,681,559; 6,335,195;6,645,489; and 6,667,064. Treated stem cells can be returned to apatient for treatment of various diseases including, but not limited to,AIDS, SCID and sickle-cell anemia.

As another example, overexpression of an oncogene may be reversed eitherby mutating the gene or by inactivating its control sequences bydeletion. Any pathology dependent upon expression of a particulargenomic sequence can be corrected or alleviated by targeted deletion ofpart or all of the sequence.

Targeted deletion can also be used to alter non-coding sequences (e.g.,regulatory sequences such as promoters, enhancers, initiators,terminators, splice sites) to alter the levels of expression of a geneproduct. Such methods can be used, for example, for therapeuticpurposes, functional genomics and/or target validation studies.

The compositions and methods described herein also allow for novelapproaches and systems to address immune reactions of a host toallogeneic grafts. In particular, a major problem faced when allogeneicstem cells (or any type of allogeneic cell) are grafted into a hostrecipient is the high risk of rejection by the host's immune system,primarily mediated through recognition of the Major HistocompatibilityComplex (MHC) on the surface of the engrafted cells. The MHC comprisesthe HLA class I protein(s) that function as heterodimers that arecomprised of a common β subunit and variable a subunits. It has beendemonstrated that tissue grafts derived from stem cells that are devoidof HLA escape the host's immune response. See, e.g., Coffman et al.(1993) J Immunol 151:425-35; Markmann et al. (1992) Transplantation54:1085-9; Koller et al. (1990) Science 248:1227-30. Using thecompositions and methods described herein, genes encoding HLA proteinsinvolved in graft rejection can be mutagenized or altered by deletion,in either their coding or regulatory sequences, so that their expressionis blocked or they express a non-functional product. For example, byinactivating the gene encoding the common β subunit gene (β2microglobulin) using the methods and compositions described herein, HLAclass I can be removed from the cells to rapidly and reliably generateHLA class I null stem cells from any donor, thereby reducing the needfor closely matched donor/recipient MHC haplotypes during stem cellgrafting.

Thus, inactivation of any gene (e.g., the β2 microglobulin gene, theCTLA4 gene) can be achieved, for example, by cleavage at two sitesfollowed by joining so as to delete the sequence between the twocleavage sites.

Targeted modification of chromatin structure, as disclosed in co-ownedInternational Patent Publication No. WO 01/83793, can be used tofacilitate the binding of fusion proteins to cellular chromatin.

EXAMPLES

The following examples show that targeted cleavage at two sites inchromosomal DNA can generate large deletions of genomic sequences,including deletion of sequences between the two cleavage sites. Targetedcleavage is accomplished, in certain embodiments, using fusion proteins(ZFNs) comprising a zinc finger DNA-binding domain and a nucleasehalf-domain.

Example 1: Design of Zinc Finger/Nuclease Half-Domain Fusion Proteins(ZFNs) Targeted to the Human CCR-5 Gene

A number of zinc finger proteins were designed to bind to sites in thehuman CCR5 gene (GenBank® Accession Number AY221093). The proteins weredesigned in pairs such that, for each pair, target sites occurred onopposite DNA strands and the near edges of the target sites wereseparated by 5 nucleotide pairs.

Table 2 shows the nucleotide sequences of the target sites for thesezinc finger domains, and the locations of the target sites within thehuman CCR-5 gene. The amino acid sequences of the recognition regions oftheir zinc finger portions are also shown.

Polynucleotides encoding fusions of the zinc finger domains shown inTable 2 to the FokI cleavage half-domain were constructed, in whichsequences encoding the zinc finger domain are upstream of sequencesencoding the cleavage half-domain, such that, in the encoded proteins,the zinc finger domain is nearest the N-terminus, and the cleavagehalf-domain is nearest the C-terminus, of the fusion protein.

TABLE 2 Zinc Finger Designs for the CCR-5 Gene² Target Name sequence¹Location³ F1 F2 F3 F4 r162b2 GATGAGGATGAC 151 to DRSNLSR TSANLSR RSDNLARTSANLSR (SEQ ID NO: 2) 162 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 3) NO: 4)NO: 5) NO: 4) r162p11 GATGAGGATGAC 151 to DRSNLSR VSSNLTS RSDNLARTSANLSR (SEQ ID NO: 2) 162 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 3) NO: 6)NO: 5) NO: 4) 168c4 AAACTGCAAAAG 168 to RSDHLSE QNANRIT RSDVLSE QRNHRTT(SEQ ID NO: 7) 179 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 8) NO: 9) NO: 10)NO: 11) 168i13 AAACTGCAAAAG 168 to RSDNLSV QKINLQV RSDVLSE QRNHRTT(SEQ ID NO: 7) 179 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 12) NO: 13)NO: 10) NO: 11) r627s1 GACAAGCAGCGG 616 to RSAHLSE RSANLSE RSANLSVDRANLSR (SEQ ID NO: 14) 627 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 15)NO: 16) NO: 17) NO: 18) 633a10 CATCTGcTACTCG 633 to RSDSLSK DNSNRIKRSAVLSE TNSNRIT (SEQ ID NO: 19) 645 (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 20) NO: 21) NO: 22) NO: 23) 633b5 CATCTGctACTCGG 633 to RSDHLSEARSTRTN RSAVLSE TNSNRIT (SEQ ID NO: 24) 646 (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 8) NO: 25) NO: 22) NO: 23) Notes: ¹Nucleotides in uppercaserepresent those present in subsites bound by individual zinc fingers;those in lowercase represent nucleotides not present in a subsite ²Thezinc finger amino acid sequencesshown above (in one-letter code)represent residues -1 through +6, with respect to the start of thealpha-helical portion of each zinc finger. Finger F1 is closest to theamino terminus of the protein. ³Numbers in this column refer tonucleotide pairs downstream from the first residue of the translationinitiation codon of the human CCR-5 gene

Example 2: Targeted Deletion in the CCR5 Gene of K562 Cells Using TwoPairs of Zinc Finger Nucleases

K562 erythroleukemia cells were cultured in RPMI medium with 10% bovineserum. At a density of approximately 1×10⁶ cells/ml, cells weretransfected with two DNA constructs, each encoding a pair of zinc fingernucleases (ZFNs), with the ZFN coding sequences separated by a 2Apeptide sequence. The first plasmid (denoted 004) encoded the r162b2 and168c4 ZFNS (see Table 2 above) which were designed to cleave between+162 and +168 (with respect to the translation start) of the human CCR-5gene; the second plasmid (denoted 043) encoded the r627s1 and 633b5 ZFNs(Table 2) which were designed to cleave between +627 and +633. Controltransfections used only one of these two plasmids, or used a plasmidencoding green fluorescent protein (GFP).

Cells were concentrated 20-fold and transfection by nucleofection, usingthe AMAXA method (solution V and program T-16 for 2 million cells per 5ug of each plasmid) following the manufacturer's protocol for K562cells. Transfection efficiency was close to 90%, as estimated byexpression of Green Fluorescent Protein as a control. Forty-eight hoursafter transfection, cells were harvested and genomic DNA was isolatedusing a Dneasy® Tissue kit (Qiagen, Valencia, Calif.), following themanufacturer's protocol. The genomic DNA (200-500 ng) was used astemplate for amplification using an AccuPrime® PCR amplification kit(Invitrogen, Carlsbad, Calif.) with the following primers:

CCR5longF: (SEQ ID NO: 26) GATGGTGCTTTCATGAATTCC and CCR5longR:(SEQ ID NO: 27) GTGTCACAAGCCCACAGATA.Amplification products were analyzed by electrophoresis on 2% agarosee-gels (Invitrogen).

Results are shown in FIG. 1. In addition to a band corresponding to theamplification product obtained from chromosomes carrying a wild-typeCCR-5 gene (present in all lanes), a lower molecular weightamplification product is obtained from cells transfected with plasmidsencoding the two ZFN pairs (lane 4). The size of this lowmolecular-weight band is consistent with removal of approximately 465nucleotide pairs from the CCR-5 sequences, which corresponds to thedistance between the two targeted cleavage sites.

To confirm that targeted deletions of the endogenous CCR-5 gene had beenobtained, amplification products were cloned into the Topo-4® vector(Invitrogen, Carlsbad, Calif.) following the manufacturer's protocol.Two classes of insert size were obtained. Plasmids containing inserts ofthe smaller size class were analyzed to determine the nucleotidesequence of their inserts. The results are shown in FIG. 2, in which arepresentative number of sequences in the region around and between thetwo targeted cleavage sites are shown. It can be seen, from thesequences obtained, that sequence alterations induced by cleavage at twocleavage sites can include deletion of some or all of the sequencebetween the two cleavage sites, and can also include deletion ofadditional sequences on one or both sides.

Analysis of genomic DNA by blotting was also conducted to provide anestimate of the frequency of deletion events resulting from targetedcleavage at two sites. Genomic DNA was isolated from K562 cells that hadbeen transfected with plasmids encoding the two nuclease pairs describedearlier in this example, or from control K562 cells that had beentransfected with a plasmid encoding green fluorescent protein. Tenmicrograms of DNA was digested with XhoI and NdeI. The digestionproducts were fractionated on an agarose gel and transferred to a nylonmembrane. The membrane was incubated with a labeled probe comprisingsequences corresponding to nucleotides −246 through +9, with respect tothe first base pair of the translation initiation codon of the humanCCR-5 gene.

The probe used in this experiment identifies a 2.8 kbp XhoI-NdeIfragment in DNA from cells transfected with the GFP-encoding plasmid,corresponding to wild-type CCR-5 sequences (FIG. 3, lane 2). In cellsexpressing the two ZFN pairs, however, a band at approximately 2.3 kbp,corresponding to deleted molecules, is also present (FIG. 3, lane 1).Quantitation of this lower molecular weight band indicated a deletionfrequency of approximately 10%.

Example 3: Targeted Deletion in the CCR5 Gene of Human T-Cells Using TwoPairs of Zinc Finger Nucleases

Human T-cells were obtained by leukopheresis from a healthy donor, the Tcells were depleted in CD8 cells, then activated for two days withPHA+IL2. Transfection was performed by electroporation, using a Maxcyteelectroporation device. Cells were transfected with two DNA constructs,each encoding a pair of zinc finger nucleases (ZFNs), with the ZFNcoding sequences separated by a 2A peptide sequence. The first plasmid(denoted 149) encoded the r162p11 and 168i13 ZFNS (see Table 2 above)which were designed to cleave between +162 and +168 (with respect to thetranslation start) of the human CCR-5 gene; the second plasmid (denoted141) encoded the r627s1 and 633a10 ZFNs (Table 2) which were designed tocleave between +627 and +633. Control transfections used a plasmidencoding green fluorescent protein (GFP).

5 million cells+20 ug of DNA (10 ug of each ZFN pair-encoding plasmid)were used per transfection. Transfected cells were collected bycentrifugation 2 days after transfection. and genomic DNA was isolatedusing a DNeasy® Tissue kit (Qiagen, Valencia, Calif.), following themanufacturer's protocol. The genomic DNA (200-500 ng) was used astemplate for amplification using an AccuPrime® PCR amplification kit(Invitrogen, Carlsbad, Calif.) with the following primers:

CCR5longF: (SEQ ID NO: 26) GATGGTGCTTTCATGAATTCC and CCR5longR:(SEQ ID NO: 27) GTGTCACAAGCCCACAGATAAmplification products were analyzed by electrophoresis on 2% agarosee-gels (Invitrogen).

Results are shown in FIG. 4. In addition to a band corresponding to theamplification product obtained from chromosomes carrying a wild-typeCCR-5 gene (lane 2 and 3 of FIG. 4), a lower molecular weightamplification product is obtained from cells transfected with plasmidsencoding the two ZFN pairs (lane 2 of FIG. 4). The size of this lowmolecular-weight band is consistent with removal of approximately 465nucleotide pairs from the CCR-5 sequences, which corresponds to thedistance between the two targeted cleavage sites.

To confirm that targeted deletions of the endogenous CCR-5 gene had beenobtained, the lower molecular weight band was excised from the gel, DNAwas eluted from the band and cloned into the Topo-4® vector (Invitrogen,Carlsbad, Calif.) following the manufacturer's protocol. Resultingplasmids were analyzed to determine the nucleotide sequence of theirinserts. The results are shown in FIG. 5, in which a representativenumber of sequences in the region around and between the two targetedcleavage sites are shown. It can be seen, from the sequences obtained,that the amplification products present in the lower band containeddeletions of approximately 430 nucleotide pairs, whose endpoints lay ator near the targeted cleavage sites.

Example 4: Targeted Deletion in the CTLA4 Gene

A number of zinc finger proteins were designed to bind to sites in thehuman CTLA4 gene (GenBank® Accession Number NM_005214). The proteinswere designed in pairs such that, for each pair, target sites occurredon opposite DNA strands. For one pair, the near edges of the targetsites were separated by 5 nucleotide pairs and, for the other pair, thenear edges of the target sites were separated by 6 nucleotide pairs.

Table 3 shows the nucleotide sequences of the target sites for thesezinc finger domains, and the locations of the target sites within thehuman CTLA4 gene. The amino acid sequences of the recognition regions oftheir zinc finger portions are also shown.

Polynucleotides encoding fusions of the zinc finger domains shown inTable 3 to the FokI cleavage half-domain were constructed, in whichsequences encoding the zinc finger domain are upstream of sequencesencoding the cleavage half-domain, such that, in the encoded proteins,the zinc finger domain is nearest the N-terminus, and the cleavagehalf-domain is nearest the C-terminus, of the fusion protein.

TABLE 3 Zinc Finger Designs for the CTLA4 Gene² Target Name sequence¹Location³ F1 F2 F3 F4 r158a ATGGCTTTATGG 147 to RSDHLSQ TSSARTN QSSDLSRRSDALTQ (SEQ ID NO: 28) 158 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 29)NO: 30) NO: 31) NO: 32) 164a GCCTTGGATTTC 164 TO TNLPLNN TSSNLSR RSDSLSADRSDLSR (SEQ ID NO: 33) 175 (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 34)NO: 35) NO: 36) NO: 37) r2902b ACCCGGaCCTCAG 2890 to RSDHLSE TSSTRKTRSDHLSE TSSDRTK (SEQ ID NO: 38) 2902 (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 8) NO: 39) NO: 8) NO: 40) 2909b GCTTCGgCAGGCT 2909 to QSSDLSRRSDNLRE RSDDLSK QSSDLRR (SEQ ID NO: 41) 2921 (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 31) NO: 42) NO: 43) NO: 44) Notes: ¹Nucleotides in uppercaserepresent those present in subsites bound by individual zinc fingers;those in lowercase represent nucleotides not present in a subsite ²Thezinc finger amino acid sequences shown above (in one-letter code)represent residues -1 through +6, with respect to the start of thealpha-helical portion of each zinc finger. Finger F1 is closest to theamino terminus of the protein. ³Numbers in this column refer tonucleotide pairs downstream from the first residue of the translationinitiation codon of the human CTLA4 gene

K562 cells were cultured and transfected as described in Example 2.Cells were transfected with four DNA constructs, each encoding one ofthe zinc finger nucleases (ZFNs) identified in Table 3. Controltransfections were conducted with a vector that did not encode a ZFN(“empty vector”).

Transfected cells were collected by centrifugation 2 days aftertransfection. and genomic DNA was isolated using a DNeasy® Tissue kit(Qiagen, Valencia, Calif.), following the manufacturer's protocol.Genomic DNA (200 ng) was used as template for amplification using anAccuPrime® PCR amplification kit (Invitrogen, Carlsbad, Calif.) withprimers that yield a 3.8 kilobase pair (kbp) amplification product froma wild-type CTLA4 gene. Amplification products were analyzed by gelelectrophoresis.

The results indicated that, in addition to a band corresponding to the3.8 kbp amplification product obtained from chromosomes carrying awild-type CTLA4 gene, a lower molecular weight amplification product ofapproximately 1 kbp was obtained from cells that were transfected withplasmids encoding the four ZFNs. The size of this low molecular-weightband is consistent with removal of approximately 2.8 kilobase pairs fromthe CTLA4 locus, which corresponds to the distance between the twotargeted cleavage sites.

Nucleotide sequence analysis of amplification products, similar to thatdescribed in Example 3, confirmed that the endpoints of the deletionslay at or near the targeted cleavage sites in the endogenous CTLA4 gene.

Similar results were obtained using a second pair of ZFNs designed tocleave between +3104 and +3111, in combination with the r158a and 164anucleases described in Table 3.

Similar results were also obtained in primary human T-cells (obtainedfrom AllCells Berkeley, Calif.) using both combinations of ZFN pairs.

Example 5: Targeted Deletion of the Human IL-2Rγ Gene

Two pairs of zinc finger/nuclease half-domain fusion proteins, designedto cleave in the third exon of the IL-2Rγ (“common gamma chain”) genehave been disclosed in parent application, U.S. Patent Publication No.2005/0064474, the disclosure of which is incorporated by reference (SeeExample 2 of that application). Two pairs of zinc finger/nucleasehalf-domain fusion proteins designed to cleave in the fifth exon of theIL-2Rγ gene are also disclosed in that application (See Examples 5 and14 of U.S. Patent Publication No. 2005/0064474, incorporated byreference).

Co-expression in cells of either of the first pair of exon 3-targetednucleases and either of the second pair of exon 5-targeted nucleases(e.g., by transfection of cells with plasmids encoding the nucleases),using methods similar to those described in the previous examples,results in cleavage events in exon 3 and exon 5 of the IL-2Rγ gene.Subsequent rejoining of DNA ends can result in loss of sequences betweenthe cleavage sites, leading to deletion of approximately 1,400nucleotide pairs of the X chromosome.

All patents, patent applications and publications mentioned herein arehereby incorporated by reference, in their entireties, for all purposes.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A method for deleting a region of interest in anendogenous gene in a plant cell, the method comprising: cleaving thegenome at first and second cleavage sites surrounding the region ofinterest using first and second pairs of artificial zinc fingernucleases that bind to first, second, third and fourth target sites inthe endogenous genome, each zinc finger nuclease comprising: a fusionprotein comprising an engineered zinc finger protein DNA-binding domainthat binds to the target site in the endogenous genome; and a FokInuclease domain, wherein each target site comprises at least 3 targetsubsites and the first cleavage site lies between the first and secondtarget sites and the second cleavage site lies between the third andfourth target sites; such that region of interest is deleted from thegenome and further wherein at least two of the target sites aremaintained in the endogenous genome following deletion of the region ofinterest.
 2. The method of claim 1, wherein the first and second targetsites are separated by 2 to 50 nucleotides.
 3. The method of claim 1,wherein the region of interest comprises at least 100 base pairs.
 4. Themethod of claim 1, wherein the region of interest comprises at least 400base pairs.
 5. The method of claim 1, wherein the region of interestcomprises at least 1000 base pairs.
 6. The method of claim 1, whereinthe plant cell is from a monocotyledonous and dicotyledonous plant. 7.The method of claim 1, wherein the plant cell is a crop plant cellselected from the group consisting of a grain crop, a fruit crop, aforage crop, a vegetable crop, an oil crop, a flowering plant crop andan experimental crop.
 8. The method of claim 1, wherein the plant cellis a seed cell.
 9. A plant generated from the plant cell produced by themethod of claim 1.